Isolated dsRNA Molecules And Methods Of Using Same For Silencing Target Molecules Of Interest

ABSTRACT

An isolated dsRNA molecule comprising an antisense RNA sequence for regulating a target gene of interest in a plant or a phytopathogen of the plant, wherein the dsRNA sequence is flanked by two complementary sites to an smRNA or smRNAs expressed in the plant and wherein the dsRNA molecule further comprises a helicase binding site positioned so as to allow unwinding of the strands of the isolated dsRNA molecule to single stranded RNA (ssRNA) and recruitment of an RNA-dependent RNA polymerase so as to amplify the dsRNA molecule in the plant cell and generate secondary siRNA products of the dsRNA sequence.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/143,748, filed Dec. 30, 2013, which claims benefit of provisionalapplications 61/748,095, filed Jan. 1, 2013, 61/748,101 filed Jan. 1,2013; 61/748,094 filed Jan. 1, 2013; 61/748,099, filed Jan. 1, 2013;61/814,888, filed Apr. 23, 2013; 61/814,892, filed Apr. 23, 2013;61/814,899, filed Apr. 23, 2013; 61/814,890, filed Apr. 23, 2013;61/908,965, filed Nov. 26, 2013; and 61/908,855, filed Nov. 26, 2013,each of which is herein incorporated by reference.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled P34097US03_SL.txt, created on Jun. 29, 2018,comprising 73,728 bytes, is incorporated herein by reference.

FIELD OF THE DISCLOSURE

The present disclosure provides for, and includes, methods andcompositions for silencing target molecules of plants and plantpathogens. Also provided are plants, plant parts and seeds having dsRNAsand methods of introducing dsRNAs into seeds.

BACKGROUND

The present disclosure, in some embodiments thereof, relates to isolateddsRNA molecules and methods of using same for silencing target moleculesof interest.

RNA interference (RNAi) has been shown effective in silencing geneexpression in a broad variety of species, including plants, with wideranging implications for cancer, inherited disease, infectious diseasein plants and animals. Studies in a variety of organisms have shown thateffectors of RNAi include dsRNA and related small interfering RNAs(siRNAs; also called “short interfering RNAs” and “silencing RNAs”).Studies have also shown in a variety of organisms that dsRNA or theirsiRNA derivatives can be used to arrest, retard or even prevent avariety of pathogens, most notably viral diseases (see, for example, PCTPatent Application Publication No. WO/2003/004649).

It has been shown in some species that RNAi mediated interferencespreads from the initial site of dsRNA delivery, producing interferencephenotypes throughout the injected animal. Recently the same spreadingeffect of dsRNA has been demonstrated in bee larva. In addition,homologs of transmembrane proteins called systemic RNA interferencedefective proteins (SID) have been detected in, for example, humans,mouse and C. elegans. It is thought that SID transmembrane channels areresponsible for endocytic uptake and spreading effect of dsRNA(Aronstein et al., J. Apic Res and Bee World, 2006; 45:20-24; see alsovan Roessel P, Brand A H., “Spreading silence with Sid,” Genome Biol.5(2):208 (2004)).

Application of RNA interference technology for insects that are plantpests and other plant pests has been suggested. Moderate RNAi-typesilencing of insect genes by feeding has been demonstrated (Turner etal., Insect Mol Biol 2006; 15:383; and Araujo et al., Insect Mol. Biol2006; 36:683). Various publications have since then focused on theincorporation of dsRNA in plants as pesticides. Such incorporationmethods can be divided into transgenic gene expression and coating suchas a seed coating.

U.S. Pat. No. 6,326,193 refers to the use of recombinant insect virusessuch as baculoviruses expressing dsRNA to silence selected insect genesfor pest control. PCT Patent Application Publication No. WO 99/32619describes the use of dsRNA for reducing crop destruction by plantpathogens or pests such as arachnids, insects, nematodes, protozoans,bacteria, or fungi. PCT Patent Application Publication No. WO2004/005485 describes RNAi sequences and transgenic plants designed tocontrol plant-parasitic nematodes.

U.S. Patent Application Publication No. 20030154508 describes pestcontrol with a dsRNA against a cation-amino acid transporter/channelprotein. PCT Patent Application Publication No. WO 02/14472 describes aninverted repeat and a sense or antisense nucleic acids for inhibitingtarget gene expression in a sucking insect. U.S. Patent ApplicationPublication No. 20030150017 describes the use of RNA moleculeshomologous or complementary to a nucleotide sequence of a plant pestsuch as nematodes and insects.

Raemakers et al. (PCT Patent Application Publication Nos. WO 2007/080127and WO 2007/080126) have disclosed transgenic plants expressing RNAi forcontrolling pest infestation by insects, nematodes, fungus and otherplant pests. Among the sequences taught are sequences targetingessential genes of insects. Waterhouse et al. (U.S. Patent ApplicationPublication No. 20060272049) and Van De Craen (U.S. Patent ApplicationPublication No. 2010068172) also disclosed transgenic plants expressingdsRNA directed to essential genes of plant insect pests, for use aspesticides and insecticides. Boukharov et al. (U.S. Patent ApplicationPublication No. 20070250947) disclosed dsRNA in transgenic plants fortargeting plant parasitic nematodes.

U.S. Patent Application Publication No. 20080022423 describes thecontrol of fungal and oomycete plant pathogens by inhibiting one or morebiological functions. The disclosure provides methods and compositionsfor such control. By feeding one or more recombinant double stranded RNAmolecules provided by the disclosure to the pathogen, a reduction indisease may be obtained through suppression of gene expression. Thedisclosure is also directed to methods for making transgenic plants thatexpress the double stranded RNA molecules, and to particularcombinations of transgenic agents for use in protecting plants frompathogen infection. Also described is a seed coating with the dsRNAanti-pathogenic compositions.

PCT Patent Application Publication No. WO 2011112570 describes a methodof regulating target endogenous gene expression in growing plants/plantorgans involving topically coating onto plants/organs, a compositioncomprising polynucleotide having sequence of specific contiguousnucleotides, and a transferring agent.

U.S. Pat. No. 8,143,480 refers to methods for knock-down of a targetgenes in plants, particularly efficient and specific methods forknock-down of a target gene in plants. This disclosure also relates tomethods for silencing endogenous plant genes or plant pathogen genes. Itfurther relates to nucleic acid constructs (DNA, RNA) which comprise anucleic acid sequence that corresponds to a target gene or fragmentthereof flanked by two complementary sites to an smRNA, e.g., a miRNA(one complementary site is on either side of the nucleic acid sequence),resulting in, for example the configuration: complementary site—nucleicacid sequence that corresponds to a target gene—complementary site.Axtell and Bartell describe siRNA biogenesis in Arabidopsis (Axtell andBartel Cell. 2006 Nov. 3; 127(3):565-77.).

It has been reported that an autonomous dsRNA sequence derived fromendovirus is found in every tissue of an infected plant and at everydevelopmental stage. Thus, in 1993 Fukuhara et al. (Plant Mol. Biol.21(6):1121-1130) identified a linear, 16 kb, dsRNA in symptomlessJaponica rice that is not found in Indica rice. The dsRNA was detectedin every tissue and at every developmental stage and its copy number wasapproximately constant (about 20 copies/cell). A sequence of about 13.2kb of the dsRNA was determined and two open reading frames (ORFs) werefound. The larger ORF (ORF B) was more than 12,351 nucleotides long andencoded a polypeptide of more than 4,117 amino acid residues having anRNA helicase-like domain followed by an RNA dependent RNAPolymerase-like domain, as characterized in subsequent works publishedas Fukuhara et al. 1995 J. Biol. Chem. 270(30):18147-18149; and Moriyamaet al. 1995 Mol. Gen. Genet. 248(3):364-369.

While not limited by theory, during RNA silencing, RNAs of about 21 to24 nucleotides (nt) in length are generated, which are incorporated intoa protein complex where they serve as guide RNAs to direct thedown-regulation of gene expression at the transcriptional orposttranscriptional level. These small interfering RNAs, small silencingRNAs, or short interfering RNAs are called “siRNAs” or “microRNAs”,depending upon their biogenesis: endogenous siRNAs derive from longdouble-stranded RNA and miRNAs derive from local hairpin structureswithin longer transcripts.

RNA silencing occurs in plants, insects, nematodes and other animals. Inaddition, new compositions (e.g., nucleic acid constructs) and methodsof achieving RNA-based silencing would be useful, and plants in whichexpression of one or more genes of interest is modulated, e.g.,inhibited, would be of great use. New compositions and rapidcost-effective methods of achieving RNA-based silencing by directlymanipulating the plant seed are highly desirable.

SUMMARY OF THE INVENTION

The present disclosure provides for, and includes, methods andcompositions for the regulation of gene expression in plants.

The present disclosure provides for, and includes, isolateddouble-stranded RNA molecules having a first RNA strand of at least oneantisense RNA sequence for regulating a target gene of interest in aplant or a phytopathogen of a plant and a first heterologous RNAsequence corresponding to a first small RNA (smRNA) expressed in a plant(e.g., a first heterologous smRNA-binding sequence for binding a firstsmRNA expressed in a plant) and a second RNA strand that is a reversecomplement of the at least one antisense RNA sequence.

The present disclosure provides for, and includes, isolateddouble-stranded RNA molecules having a first RNA strand of at least oneantisense RNA sequence for regulating a target gene of interest in aplant or a phytopathogen of a plant, a first heterologous RNA sequencecorresponding to a first small RNA (smRNA) expressed in the plant (e.g.,a first heterologous smRNA-binding sequence for binding a first smRNAexpressed in a plant), a helicase binding sequence and a second RNAstrand that is a reverse complement of the at least one antisense RNAsequence.

The present disclosure provides for, and includes, isolateddouble-stranded RNA molecules having a first RNA strand of at least oneantisense RNA sequence for regulating a target gene of interest in aplant or a phytopathogen of a plant and a first heterologous RNAsequence corresponding to a first small RNA (smRNA) expressed in theplant (e.g., a first heterologous smRNA-binding sequence for binding afirst smRNA expressed in a plant) and a second RNA strand that is areverse complement of the at least one antisense RNA sequence and thefirst heterologous RNA sequence.

The present disclosure provides for, and includes, isolateddouble-stranded RNA molecules having a first RNA strand of at least oneantisense RNA sequence for regulating a target gene of interest in aplant or a phytopathogen of a plant, a first heterologous RNA sequencecorresponding to a first small RNA (smRNA) expressed in a plant (e.g., afirst heterologous smRNA-binding sequence for binding a first smRNAexpressed in a plant), a helicase binding sequence and a second RNAstrand that is a reverse complement of the at least one antisense RNAsequence and the first heterologous RNA sequence.

The present disclosure provides for, and includes, isolateddouble-stranded RNA molecules having a first RNA strand of at least oneantisense RNA sequence for regulating a target gene of interest in aplant or a phytopathogen of a plant, a first heterologous RNA sequencecorresponding to a first small RNA (smRNA) expressed in said plant(e.g., a first heterologous smRNA-binding sequence for binding a firstsmRNA expressed in a plant), a helicase binding sequence and a secondRNA strand that is a reverse complement of the at least one antisenseRNA sequence and the first heterologous RNA sequence.

The present disclosure provides for, and includes, isolateddouble-stranded RNA molecules having a first RNA strand of at least oneantisense RNA sequence for regulating a target gene of interest in aplant or a phytopathogen of a plant, a first heterologous RNA sequencecorresponding to a first small RNA (smRNA) expressed in said plant(e.g., a first heterologous smRNA-binding sequence for binding a firstsmRNA expressed in a plant), a helicase binding sequence and a secondRNA strand that is a reverse complement of the at least one antisenseRNA sequence, the first heterologous RNA sequence, and helicase bindingsequence.

The present disclosure provides for, and includes, isolateddouble-stranded RNA molecules having a first RNA strand of at least oneantisense RNA sequence for regulating a target gene of interest in aplant or a phytopathogen of a plant, a first heterologous RNA sequencecorresponding to a first small RNA (smRNA) expressed in the plant (e.g.,a first heterologous smRNA-binding sequence for binding a first smRNAexpressed in a plant), a second heterologous RNA sequence correspondingto a second smRNA expressed in the plant (e.g., a second heterologoussmRNA-binding sequence for binding a second smRNA expressed in a plant),where the first heterologous smRNA and said second heterologous smRNAflank the at least one antisense RNA sequence, and a second RNA strandthat is a reverse complement of the at least one antisense RNA sequence.

The present disclosure provides for, and includes, isolateddouble-stranded RNA molecules having a first RNA strand of at least oneantisense RNA sequence for regulating a target gene of interest in aplant or a phytopathogen of a plant, a first heterologous RNA sequencecorresponding to a first small RNA (smRNA) expressed in the plant (e.g.,a first heterologous smRNA-binding sequence for binding a first smRNAexpressed in a plant), a second heterologous RNA sequence correspondingto a second smRNA expressed in the plant (e.g., a second heterologoussmRNA-binding sequence for binding a second smRNA expressed in a plant),where the first heterologous smRNA and said second heterologous smRNAflank the at least one antisense RNA sequence, and a second RNA strandthat is a reverse complement of the at least one antisense RNA sequence,and first heterologous RNA sequence.

The present disclosure provides for, and includes, isolateddouble-stranded RNA molecules having a first RNA strand of at least oneantisense RNA sequence for regulating a target gene of interest in aplant or a phytopathogen of a plant, a first heterologous RNA sequencecorresponding to a first small RNA (smRNA) expressed in the plant (e.g.,a first heterologous smRNA-binding sequence for binding a first smRNAexpressed in a plant), a second heterologous RNA sequence correspondingto a second smRNA expressed in the plant (e.g., a second heterologoussmRNA-binding sequence for binding a second smRNA expressed in a plant),where the first heterologous smRNA and said second heterologous smRNAflank the at least one antisense RNA sequence, and a second RNA strandthat is a reverse complement of the at least one antisense RNA sequence,first heterologous RNA sequence, and second heterologous RNA sequence.

The present disclosure provides for, and includes, isolateddouble-stranded RNA molecules having a first RNA strand of at least oneantisense RNA sequence for regulating a target gene of interest in aplant or a phytopathogen of a plant, a helicase binding sequence, afirst heterologous RNA sequence corresponding to a first small RNA(smRNA) expressed in the plant (e.g., a first heterologous smRNA-bindingsequence for binding a first smRNA expressed in a plant), a secondheterologous RNA sequence corresponding to a second smRNA expressed inthe plant (e.g., a second heterologous smRNA-binding sequence forbinding a second smRNA expressed in a plant), where the firstheterologous smRNA and said second heterologous smRNA flank the at leastone antisense RNA sequence, and a second RNA strand that is a reversecomplement of the at least one antisense RNA sequence, firstheterologous RNA sequence, and second heterologous RNA sequence.

According to some embodiments of the present disclosure there isprovided an isolated dsRNA molecule comprising an antisense RNA sequencefor regulating a target gene of interest in a plant or a phytopathogenof the plant, wherein the dsRNA sequence is flanked by two complementarysites to an smRNA or smRNAs expressed in the plant and wherein the dsRNAmolecule further comprises a helicase binding site positioned so as toallow unwinding of the strands of the isolated dsRNA molecule to singlestranded RNA (ssRNA) and recruitment of an RNA-dependent RNA Polymeraseso as to amplify the dsRNA molecule in the plant cell and generatesecondary siRNA products of the dsRNA sequence.

According to some embodiments of the present disclosure there isprovided an isolated dsRNA molecule comprising an antisense RNA sequencefor regulating a target gene of interest in a plant or a phytopathogenof the plant, wherein the dsRNA sequence is flanked by two complementarysites to an smRNA or smRNAs expressed in the plant.

According to an embodiment of some embodiments of the present disclosurethere is provided an isolated dsRNA molecule comprising an antisense RNAsequence for regulating a target gene of interest in a plant or aphytopathogen of the plant, wherein the dsRNA molecule further comprisesa complementary site to an smRNA expressed in the plant located upstreamor downstream the dsRNA.

According to some embodiments of the disclosure, the isolated dsRNAmolecule further comprises a helicase binding site positioned so as toallow unwinding of the strands of the isolated dsRNA molecule to singlestranded RNA (ssRNA) and recruitment of an RNA-dependent RNA Polymeraseso as to amplify the dsRNA molecule in the plant cell.

According to some embodiments of the disclosure, the complementary siteto the smRNA is located downstream of the dsRNA sequence.

According to some embodiments of the disclosure, the complementary siteto the smRNA is located upstream of the dsRNA sequence.

According to some embodiments of the disclosure, one of the twocomplementary sites to the smRNA or smRNAs comprises a mutationrendering it resistant to cleavage by the complementary smRNA.

According to some embodiments of the disclosure, the helicase bindingsite is positioned upstream of the dsRNA sequence.

According to some embodiments of the disclosure, wherein the helicasebinding site is position in the dsRNA sequence for regulating a targetgene of interest in the plant or the phytopathogen of the plant.

According to some embodiments of the disclosure, the helicase bindingsite is positioned upstream of the dsRNA sequence and the twocomplementary sites to the smRNA or smRNAs flank the helicase bindingsite.

According to some embodiments of the disclosure, the smRNA or smRNAs isselected from the group consisting of a miRNA and a siRNA.

According to some embodiments of the disclosure, the smRNA or smRNAs isa miRNA.

According to some embodiments of the disclosure, the miRNA is smRNA390.

According to some embodiments of the disclosure, the plant comprises aTAS locus that has a second smRNA complementary site.

According to some embodiments of the disclosure, the first and secondcomplementary sites are naturally found flanking the TAS locus in theplant.

According to some embodiments of the disclosure, the smRNA is an smRNAfor which complementary sites are naturally found flanking a TAS locusin a plant.

According to some embodiments of the disclosure, the two complementarysites are complementary sites for the same smRNA.

According to some embodiments of the disclosure, the two complementarysites comprise difference sequences.

According to some embodiments of the disclosure, the two complementarysites comprise the same sequence.

According to some embodiments of the disclosure, the smRNAs arenon-identical.

According to some embodiments of the disclosure, the smRNA or smRNAs isselected from the group consisting of miR390, miR161.1, miR168, miR393,miR828 and miR173. According to some embodiments of the disclosure, theplant is a crop plant.

According to an embodiment of some embodiments of the present disclosurethere is provided a method of silencing expression of a target gene ofinterest in a plant, the method comprising introducing the isolateddsRNA molecule, and wherein the dsRNA sequence is for silencing thetarget gene of interest in the plant, thereby silencing expression ofthe target gene of interest in the plant.

According to an embodiment of some embodiments of the present disclosurethere is provided a method of introducing dsRNA molecule into a seed,the method comprising contacting the seed with the isolated dsRNAmolecule under conditions which allow penetration of the dsRNA moleculeinto the seed, thereby introducing the dsRNA molecule into the seed.

According to an embodiment of some embodiments of the present disclosurethere is provided an isolated seed comprising the isolated dsRNAmolecule.

According to some embodiments of the disclosure, the isolated seed isdevoid of a heterologous promoter for driving expression of the dsRNAmolecule in the plant.

According to an embodiment of some embodiments of the present disclosurethere is provided a seed comprising the isolated dsRNA molecule and thesecondary siRNA products.

According to an embodiment of some embodiments of the present disclosurethere is provided a plant or plant part generated from the seed.

According to an embodiment of some embodiments of the present disclosurethere is provided a seed containing device comprising a plurality of theseeds.

According to an embodiment of some embodiments of the present disclosurethere is provided a sown field comprising a plurality of the seeds.

According to an embodiment of some embodiments of the present disclosurethere is provided a method of producing a plant the method comprising:(a) providing the seed; and (b) germinating the seed so as to producethe plant.

According to an embodiment of some embodiments of the present disclosurethere is provided a method of modulating gene expression in a plant, themethod comprising: (a) contacting a seed of the plant with the dsRNAmolecule, under conditions which allow penetration of the dsRNA moleculeinto the seed, thereby introducing the dsRNA molecule into the seed; andoptionally (b) generating a plant of the seed.

According to some embodiments of the disclosure, the penetration is toan endosperm and alternatively or additionally an embryo of the seed.

According to an embodiment of some embodiments of the present disclosurethere is provided a method of silencing expression of a target gene in aphytopathogenic organism, the method comprising providing to thephytopathogenic organism the plant or plant part, thereby silencingexpression of a target gene in the phytopathogenic organism.

According to some embodiments of the disclosure, the phytopathogenicorganism is selected from the group consisting of a fungus, a nematode,an insect, a bacteria and a virus.

According to an embodiment of some embodiments of the present disclosurethere is provided a kit for introducing a dsRNA molecule to seedscomprising; (i) the dsRNA molecule; and (ii) a priming solution.

According to some embodiments of the disclosure, the dsRNA molecule andthe priming solution are comprised in separate containers.

According to an embodiment of some embodiments of the present disclosurethere is provided a pesticidal composition comprising the isolated dsRNAmolecule.

According to some embodiments of the disclosure, the contacting iseffected by inoculating the seed with the dsRNA molecule.

According to some embodiments of the disclosure, the method furthercomprises priming the seed prior to the contacting.

According to some embodiments of the disclosure, the priming is effectedby: (i) washing the seed prior to the contacting; and (ii) drying theseed following step (i).

According to some embodiments of the disclosure, the washing is effectedin the presence of double deionized water.

According to some embodiments of the disclosure, the washing is effectedfor 2-6 hours. According to some embodiments of the disclosure, thewashing is effected at 4-28° C. According to some embodiments of thedisclosure, the drying is effected at 25-30° C. for 10-16 hours.

According to some embodiments of the disclosure, the contacting iseffected in a presence of the dsRNA molecule at a final concentration of0.1-100 μg/μl.

According to some embodiments of the disclosure, the contacting iseffected in a presence of the dsRNA molecule at a final concentration of0.1-0.5 μg/μl.

According to some embodiments of the disclosure, the method furthercomprises treating the seed with an agent selected from the groupconsisting of a pesticide, a fungicide, an insecticide, a fertilizer, acoating agent and a coloring agent following the contacting.

According to some embodiments of the disclosure, the treating comprisescoating the seed with the agent.

According to some embodiments of the disclosure, the conditions allowaccumulation of the dsRNA molecule in the endosperm and alternatively oradditionally embryo of the seed.

According to some embodiments of the disclosure, a concentration of thedsRNA molecule is adjusted according to a parameter selected from thegroup consisting of, seed size, seed weight, seed volume, seed surfacearea, seed density and seed permeability.

According to some embodiments of the disclosure, the contacting iseffected prior to breaking of seed dormancy and embryo emergence.

According to some embodiments of the disclosure, the seed is a primedseed.

According to some embodiments of the disclosure, the seed comprises RNAdependent RNA polymerase activity for amplifying expression of the dsRNAmolecule.

According to some embodiments of the disclosure, the seed is a hybridseed.

According to an aspect of some embodiments of the present disclosurethere is provided an isolated dsRNA molecule comprising a nucleic acidsequence which comprises in a sequential order from 5′ to 3′, anendovirus 5′ UTR, an endovirus RNA Dependent RNA Polymerase (RDRP)coding sequence, an endovirus 3′ UTR and a multiple cloning site flankedby the RDRP and the 3′ UTR.

According to an aspect of some embodiments of the present disclosurethere is provided an isolated dsRNA molecule comprising a nucleic acidsequence which comprises in a sequential order from 5′ to 3′, anendovirus 5′ UTR, an endovirus RNA Dependent RNA Polymerase (RDRP)coding sequence, an endovirus 3′ UTR and a nucleic acid sequence forregulating a target gene flanked by the RDRP and the 3′ UTR.

According to some embodiments of the disclosure, the endovirus 5′ UTR,endovirus RNA Dependent RNA Polymerase (RDRP) coding sequence and theendovirus 3′ UTR are selected capable of autonomous replication in theplant cell.

According to some embodiments of the disclosure, the 5′ UTR is as setforth in SEQ ID NO: 14.

According to some embodiments of the disclosure, the 3′ UTR is as setforth in SEQ ID NO: 22.

According to some embodiments of the disclosure, the endovirus RNADependent RNA Polymerase (RDRP) coding sequence is as set forth in SEQID NO: 23.

According to some embodiments of the disclosure, the nucleic acidsequence for regulating a target gene is 17-600 bp long.

According to some embodiments of the disclosure, the nucleic acidsequence for regulating a target gene is selected from the groupconsisting of a miRNA and a siRNA.

According to some embodiments of the disclosure, the nucleic acidsequence for regulating a target gene is a miRNA.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the disclosure pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the disclosure, examples ofmethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the disclosure are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the disclosure. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the disclosure may be practiced.

FIG. 1 presents fluorescent images of siGLO-treatment rice seeds over a24 hour period according to embodiment of the present disclosure.

FIG. 2A presents a schematic representation of the Solanum Lycopersicum(Lycopersicon esculentum) TAS3 gene according to an embodiment of thepresent disclosure. Mir390BS is displayed in a darker gray box (SEQ IDNO: 319). The light gray box represents the 5′ Mut Mir390BS (SEQ ID NO:319).

FIG. 2B presents a schematic representation of dsRNA construct #1 havingan exogenous trigger control according to an embodiment of the presentdisclosure. The construct includes a 234 bp exogenous sequence providedin Table 5 (e.g., Trigger #1).

FIG. 2C presents a schematic representation of dsRNA construct #2 havinga dual Mir390BS sequence on the sense strand and an exogenous sequenceaccording to an embodiment of the present disclosure. The construct iscomprises 3 parts from 5′ to 3′: a 5′ Mut Mir390BS sequence, a 234 bpexogenous sequence in reverse complement orientation, and a 3′ Mir390BS.The sequences are presented in Table 5 (e.g., Trigger #2).

FIG. 3 presents a schematic representation of dsRNA construct#3 having adual Mir390BS on both on the sense and antisense strands. The constructis composed of 5 parts from 5′ to 3′: 3′ Mir390BS in the reversecomplement orientation, 5′ Mut Mir390BS, a 234 bp exogenous sequence inthe reverse complement orientation, 5′ Mut Mir390BS in the reversecomplement orientation and 3′ Mir390BS. For sequence, see Table 5(Trigger #3).

FIG. 4 presents a schematic representation of dsRNA construct #4 havingmiR390S as overhangs. This construct is composed of two differentstrands. The sense strand is composed of 3 parts from 5′ to 3′: 5′ MutMir390BS, a 234 bp exogenous sequence in the reverse complementorientation, 3′ Mir390BS. The antisense is composed of only one part: a234 bp exogenous sequence in the sense orientation. For sequences, seeTable 5 (Sense-Trigger#4, Antisense-Trigger #5).

FIG. 5 presents a schematic representation of dsRNA construct #5 havingmiR390BS and a helicase binding sequence (Helicase BS). This constructis composed of 4 parts from 5′ to 3′: 5′ Mut Mir390BS, a 234 bpexogenous sequence in the reverse complement orientation, Helicase BS inthe reverse complement orientation, 3′ Mir390BS. For sequence see Table5 (Trigger #6).

FIG. 6 presents a schematic representation of dsRNA construct #6 havingMir390BS on both strands and Helicase BS as an overhang. This constructis composed of two different strands. The sense strand is composed of 5parts from 5′ to 3: ‘3’ Mir390BS in the reverse complement orientation,5′ Mut Mir390BS, a 234 bp exogenous sequence in the reverse complementorientation, 5′ Mut Mir390BS in the reverse complement orientation, and3′Mir390BS. The antisense is composed of 6 parts from 5′ to 3′:3′Mir390BS in the reverse complement orientation, 5′ Mut Mir390BS, a 234bp exogenous sequence in the sense orientation, 5′ Mut Mir390BS in thereverse complement orientation, 3′ Mir390BS and an Helicase BS as anoverhang. For sequences, see Table 5 (Sense-Trigger #7,Antisense-Trigger #8).

FIG. 7 presents a schematic representation of dsRNA construct #7 havingSense dual Mir390BS coupled with Antisense Mir4376BS. This construct iscomposed of 5 parts from 5′ to 3′: 5′ Mut Mir390BS, a 234 bp exogenoussequence in the reverse complement orientation, Mir4376BS in the reversecomplement orientation and 3′ Mir390BS. For sequence, see Table 5(Trigger #9).

FIG. 8 presents a schematic representation of dsRNA construct #8 anEndogenous Trigger Control. This construct is composed of one part: a234 bp of the endogenous TAS3 sequence. For sequence, see Table 5(Trigger #10).

FIG. 9 presents a schematic representation of dsRNA construct#9—Mir390BS+Endogenous insert. This construct is composed of 3 partsfrom 5′ to 3′: 5′ Mut Mir390BS, a 234 bp of the endogenous TAS3 sequenceand 3′ Mir390BS. For sequence, see Table 5 (Trigger #11).

FIGS. 10A-E are schematic representations of dsRNA constructs of thepresent disclosure.

FIGS. 11A-B present graphs showing real-time PCR analyses of ARF3 andARF4 mRNA expression in roots 14 days after seed treatment according toan embodiment of the present disclosure.

FIGS. 11C-E present graphs showing the results of real-time PCR analysesof GFP in seedlings seven days after seed treatment according to anembodiment of the present disclosure.

FIG. 11F presents a graph showing the results of real-time PCR analysesof GFP in leaves 30 days after seed treatment according to an embodimentof the present disclosure.

FIGS. 12A-E present graphs showing the results of real-time PCR analysesof GFP in shoots seven days after seed treatment according to anembodiment of the present disclosure.

FIGS. 13A-B presents graphs showing the results of real-time PCRanalyses of GFP in shoots 14 days after seed treatment according to anembodiment of the present disclosure.

FIGS. 14A-B presents graphs showing the results of real-time PCRanalyses of GFP in shoots seven days (A) and 14 days (B), according toan embodiment of the present disclosure.

FIGS. 15A-B presents graphs showing the results of real-time PCRanalyses of GUS in shoots seven days (A) and 14 days (B) after seedtreatment according to an embodiment of the present disclosure.

DETAILED DESCRIPTION

The present disclosure, in some embodiments thereof, relates to andprovides for isolated dsRNA molecules and methods of using same forsilencing target molecules of interest.

The present disclosure further includes and provides for compositionsand methods for silencing gene expression.

The present disclosure provides for, and includes tools for overcomingthe delivery obstacle and amplifying the small interfering RNA (siRNA)levels within the plant cell to thereby efficiently down-regulate targetgenes of interest.

Before explaining at least one embodiment of the disclosure in detail,it is to be understood that the disclosure is not necessarily limited inits application to the details set forth in the following description orexemplified by the Examples. The disclosure is capable of otherembodiments or of being practiced or carried out in various ways.

It is understood that any Sequence Identification Number (SEQ ID NO)disclosed in the instant application can refer to either a DNA sequenceor a RNA sequence, depending on the context where that SEQ ID NO ismentioned, even if that SEQ ID NO is expressed only in a DNA sequenceformat or a RNA sequence format. For example, SEQ ID NO: 1 is expressedin a DNA sequence format (e.g., reciting T for thymine), but it canrefer to either a DNA sequence that corresponds to a T7 DNA DependentRNA Polymerase primer nucleic acid sequence, or the RNA sequence of anRNA molecule nucleic acid sequence. Similarly, though SEQ ID NO: 25 isexpressed in a RNA sequence format (e.g., reciting U for uracil),depending on the actual type of molecule being described, SEQ ID NO: 25can refer to either the sequence of a RNA molecule comprising a dsRNA,or the sequence of a DNA molecule that corresponds to the RNA sequenceshown. In any event, both DNA and RNA molecules having the sequencesdisclosed with any substitutes are envisioned.

As used herein, the terms “homology” and “identity” when used inrelation to nucleic acids, describe the degree of similarity between twoor more nucleotide sequences. The percentage of “sequence identity”between two sequences is determined by comparing two optimally alignedsequences over a comparison window, such that the portion of thesequence in the comparison window may comprise additions or deletions(gaps) as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whichthe identical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity. A sequence that is identical at every position incomparison to a reference sequence is said to be identical to thereference sequence and vice-versa. An alignment of two or more sequencesmay be performed using any suitable computer program. For example, awidely used and accepted computer program for performing sequencealignments is CLUSTALW v1.6 (Thompson, et al. Nucl. Acids Res., 22:4673-4680, 1994).

Homologous sequences include both orthologous and paralogous sequences.The term “paralogous” paralogous” relates to gene-duplications withinthe genome of a species leading to paralogous genes. The term“orthologous” relates to homologous genes in different organisms due toancestral relationship. For instance in this case, other plant RNAviruses.

Identity (e.g., percent homology) can be determined using any homologycomparison software, including for example, the BlastN software of theNational Center of Biotechnology Information (NCBI) such as by usingdefault parameters.

According to some embodiments of the disclosure, the identity is aglobal identity, i.e., an identity over the entire nucleic acidsequences of the disclosure and not over portions thereof.

The degree of homology or identity between two or more sequences can bedetermined using various known sequence comparison tools. Following is anon-limiting description of such tools which can be used along with someembodiments of the disclosure.

As used herein, the terms “exogenous polynucleotide” and “exogenousnucleic acid molecule” relative to an organism refer to a heterologousnucleic acid sequence which is not naturally expressed within thatorganism, for example a plant. An exogenous nucleic acid molecule may beintroduced into an organism in a stable or transient manner. Anexogenous nucleic acid molecule may comprise a nucleic acid sequencewhich is identical or partially homologous to an endogenous nucleic acidsequence of the organism. In certain embodiments, an “exogenouspolynucleotide” and “exogenous nucleic acid molecule” may refer to anucleic acid sequence expressed or present in a plant, eithertransiently or stably. As used herein, the terms “endogenouspolynucleotide” and “endogenous nucleic acid” refers to nucleic acidsequences that are found in an organism's cell. In certain aspects, anendogenous nucleic acid may be part of the nuclear genome or the plastidgenome. In other aspects, an endogenous nucleic acid may be foundoutside the nuclear or plastid genomes. As used herein, endogenousnucleic acids do not include viral, parasite or pathogen nucleic acids,for example an endovirus sequence. The present disclosure provides for,and includes, compositions comprising exogenous polynucleotides andexogenous nucleic acid molecules and methods for introducing them into atarget organism. The present disclosure provides for, and includes,compositions comprising exogenous polynucleotides and exogenous nucleicacid molecules in combination with endogenous nucleic acids andpolynucleotides and methods for introducing them into a target organism.The present disclosure provides for, and includes, compositionscomprising recombinant endogenous nucleic acids and polynucleotides andmethods for introducing them into a target organism.

The present disclosure provides for, and includes dsRNA molecules whichare processed through the trans-acting siRNA (ta-siRNA) pathway.Transacting siRNAs are a subclass of siRNAs that function like miRNAs torepress expression of target genes. While not limited to any particulartheory, trans-acting siRNAs form by transcription of ta-siRNA-generatinggenes found at trans-acting (TAS) loci. A ta-siRNA precursor is anynucleic acid molecule, including single-stranded or double-stranded DNAor RNA, that can be transcribed and/or processed to release a ta-siRNA.Cleavage of the primary transcript occurs through a guided RISCmechanism, conversion of one of the cleavage products to dsRNA, andprocessing of the dsRNA by dicer or dicer-like (DCL) enzymes. While notlimited by any particular theory, it is thought that RNA-dependent RNApolymerase 6 (RDR6) (or related enzymes) function in posttranscriptionalRNAi of sense transgenes, some viruses, and specific endogenous mRNAsthat are targeted by trans-acting siRNAs (ta-siRNAs) (see Dalmay et al.,Cell 101:543-553, 2000; Mourrain et al., Cell 101:533-542, 2000;Peragine et al., Genes & Dev 18:2369-2379, 2004; Vazquez et al., MolCell 16:69-79, 2004b; Yu et al., Mol Plant Microbe Interact 16:206-216,2003). Again, while not being limited to any particular theory, it isthought that ta-siRNAs arise from transcripts that are recognized byRDR6, in cooperation with SGS3, as a substrate to form dsRNA. The dsRNAis processed accurately in 21-nucleotide steps by DCL1 to yield a set of“phased” ta-siRNAs. These ta-siRNAs interact with target mRNAs to guidecleavage by the same mechanism as do plant miRNAs (Peragine et al.,Genes & Dev 18:2369-2379, 2004; Vazquez et al., Mol Cell 16:69-79, 2004;Allen et al., Cell 121:207-221, 2005). Trans-acting siRNAs are conservedamong distantly related plant species and have been maintained over along evolutionary period. The design and construction of ta-siRNAconstructs and their use in the modulation of protein in transgenicplant cells is disclosed by Allen and Carrington in US PatentApplication Publication US 2006/0174380 A1 (now U.S. Pat. No. 8,030,473)which is incorporated herein by reference.

As used herein, the term “dsRNA sequence” refers to, and includes, adouble-stranded sequences having a first strand and a second strand thatis a reverse complement of the first strand. It will be understood thatreference to an antisense RNA sequence for regulating a target gene ofinterest and a sense RNA sequence for regulating a target gene ofinterest, would necessarily include a dsRNA sequences when included in adsRNA molecule. For clarity, the sequences for targeting a gene ofinterest for regulation will be generally referenced as the antisenseRNA sequence and provides for a standard reference point for the 5′ and3′ ends. As used herein, the ‘antisense strand’ refers to the strandhaving the antisense RNA sequence for regulating (e.g., suppressing orsilencing) a target gene of interest. One of ordinary skill in the artwould further understand that reference to a single strand, whether thesense or antisense strand, provides a definition and sequence for thereverse complement strand. Further, it is well understood that a singlenucleic acid strand and its reverse complement provide for adouble-stranded nucleic acid. One of ordinary skill in the art wouldunderstand that an RNA and DNA sequence may be readily substituted usingthe well-known base pairing rules and as provided above. One of ordinaryskill in the art would further understand that binding can occur betweentwo polynucleotide sequences that are characterized by having sufficientsequence complementarity (which need not be 100% complementarity) toallow hybridization between the two polynucleotides (e.g., binding orhybridization under common physiological conditions). Thus, a“heterologous smRNA-binding sequence for binding a first small RNA” neednot be 100% complementary to the sequence of the first small RNA (forexample, where the heterologous smRNA-binding sequence is complementaryto the sequence of the first small RNA except for one or more mutationsor mismatches at the site where cleavage mediated by the small RNA wouldnormally occur), although in some embodiments the complementarity is100%. The present disclosure provides for, and includes, an isolateddsRNA molecule comprising an antisense RNA sequence for regulating atarget gene of interest in a plant or a phytopathogen of the plant,wherein the dsRNA sequence is flanked by two complementary sites to ansmRNA expressed in the plant. In some embodiments, the dsRNA sequencemay be flanked by two complementary sites from the same smRNA expressedin the plant. In other embodiments, the dsRNA sequence may be flanked bycomplementary sites from two different smRNAs. In yet other embodiments,the dsRNA sequence may be flanked by four complementary sitescorresponding to one or more smRNAs expressed in a plant (e.g., twoheterologous sequences on one side and two heterologous sequences on theother side of the dsRNA sequence). In certain embodiments, the dsRNAmolecule further comprises a helicase binding site positioned so as toallow unwinding of the strands of the isolated dsRNA molecule to singlestranded RNA (ssRNA) and amplification by recruitment of anRNA-dependent RNA Polymerase (RDRP) when introduced into a host cell. Inother embodiments, the helicase and other proteins may be provided invitro, for example as part of a cell extract. Methods of in vitroanalysis are known in the art. In certain embodiments, the host cell isa plant cell. In some embodiments, introduction of the dsRNA moleculeinto a plant cell results in the recruitment of a helicase and RDRP andthe generation of secondary siRNA products corresponding to the dsRNAsequence for regulating a target gene of interest in a plant or aphytopathogen of the plant. In certain embodiments, the target gene issilenced. In other embodiments, expression of the target gene isenhanced.

According to another embodiment of the disclosure there is provided, andincluded, an isolated dsRNA molecule comprising an antisense RNAsequence for regulating a target gene of interest in a plant or aphytopathogen of the plant, wherein the dsRNA sequence is flanked by twocomplementary sites to an smRNA or smRNAs expressed in the plant. Incertain embodiments, the target gene is silenced. In other embodiments,expression of the target gene is enhanced.

According to a further embodiment of the disclosure there is provided,and included, an isolated dsRNA molecule comprising an antisense RNAsequence for regulating a target gene of interest in a plant or aphytopathogen of the plant, wherein the dsRNA molecule further comprisesa complementary site to an smRNA expressed in the plant located upstreamor downstream the dsRNA. In certain embodiments, the target gene issilenced. In other embodiments, expression of the target gene isenhanced.

Not to be limited by theory, a possible downstream mechanism for a dsRNAconstruct of the present disclosure having two flanking heterologous RNAsequences corresponding to an smRNA, with one sequence being anon-cleavable mutant (for example, mir390 BS and Mir390 Mut BS of FIG.2C) includes unwinding of the dsRNA in the cell. Following the openingof the double stranded RNA into two single strands the sense strand mayrecruit an AGO7, or AGO7-like, protein to the flanking heterologous RNAsequences. This binding, in turn, may lead to cleavage at a thenon-mutated heterologous RNA sequence (for example Mir390 BS of FIG. 2C)and localization of this single stranded RNA inside a cytoplasmicprocessing center (Evidence for such a processing center was reported inKumakura et al. (2009). SGS3 and RDR6 interact and colocalize incytoplasmic SGS3/RDR6-bodies. (2009). FEBS Letters, 583, 1261-1266 andJouannet et al. (2012). Cytoplasmic Arabidopsis AGO7 accumulates inmembrane-associated siRNA bodies and is required for to-siRNAbiogenesis. EMBO Journal, 31, 17041713.). The antisense strand may theneither be diced or cleaved but is not expected to take part inadditional amplification of the exogenous sequence (e.g., the gene ofinterest of FIG. 2C).

Not to be limited by theory, an alternative downstream mechanism fordsRNA construct of the present disclosure having two flankingheterologous RNA sequences corresponding to an smRNA (for example, asprovided in FIG. 2C) similarly starts with the unwinding of the dsRNA ina cell. In this non-limiting theoretical mechanism, the sense strand istranslocated to a processing center that may have an accumulation of aRNA Dependent RNA Polymerase (RDRP) that is predicted to lead to theformation of antisense transcripts. Preferably, each template of senseRNA will serve for multiple rounds of antisense RNA production.Following antisense RNA accumulation, it may be that the merelocalization of this transcript inside the processing center enablesRDRP recruitment and creation of double-stranded RNAs (even though thisstrand may lack a recognizable element of the TAS system). Some of thesedouble stranded RNAs may be translocated to the nucleus where to bediced into ta-siRNAs against an exogenous sequence and some of thedouble stranded RNA may remain in the processing center where it willunwind again and lead to further cycles of amplification. One possiblemediator of the unwinding process inside the processing center is theSDE3 RNA helicase (see Garcia et al. (2012). Ago Hook and RNA HelicaseMotifs Underpin Dual Roles for SDE3 in Antiviral Defense and Silencingof Nonconserved Intergenic Regions. Mol Cell, 48, 109-120.).

Not to be limited by theory, a possible downstream mechanism for dsRNAconstruct #3 (FIG. 3) also begins with unwinding of the dsRNA into twosingle strands.

Focusing on the outcome of the sense strand, it may be recognized byMir390-Ago7 at both Mir390 Binding sites. The binding of this complexmay lead to cleavage at the 3′ Mir390BS and to the translocation of thistruncated transcript into a processing center. Inside the processingcenter it may serve as a template for the creation of multipletranscripts of antisense strands. The newly created antisense strandsmay contain recognizable Mir390 binding sites and therefore may be ableto recruit Ago7 and Mir390 to the 5′ Mut Mir390BS. This binding is mayenable efficient recruitment of RDRP and creation of double strandedRNAs. Some of this double stranded RNA may be translocated to thenucleus and diced into ta-siRNAs whereas other dsRNA may be expected tocontinue to additional rounds of unwinding and amplification.

Not to be limited by theory, in the alternative, dsRNA construct #3 maybe unwound in the cell to a ssRNA. Focusing on the outcome of theantisense strand, it may be recognized by Mir390-Ago7 at both Mir390Binding sites. The binding of this complex may lead to cleavage at the3′ Mir390BS and to the translocation of this truncated transcript intothe processing center. Inside the processing center it may serve as atemplate for the creation of multiple transcripts of sense strands. Thenewly created sense strands will contain recognizable Mir390 bindingsites and therefore may be able to recruit Ago7 and Mir390 to the 5′ MutMir390BS. This binding may enable efficient recruitment of RDRP andcreation of double stranded RNAs. Some of this double stranded RNA maybe translocated to the nucleus and diced into ta-siRNAs whereas otherdsRNA may continue to additional rounds of unwinding and amplification.

Not to be limited by theory, in another alternative mechanism, dsRNAconstruct #3 is undergoes strand unwinding in a cell. Newly synthesizedsense and antisense strands (possibly resulting from the mechanismsdescribed above may serve as templates for multiple rounds of RDRPrecruitment and dsRNA amplification. This construct may lead to anoptimal amplification due to the presence of the 5′ Mut Mir390BS on bothstrands enabling ongoing recruitment of Mir390-Ago7 complex.

As used herein, the term “upstream” refers to positions that are 5′ endof the polynucleotide. In certain aspects, upstream refers to the 5′location of sequences relative to an antisense sequence for regulating atarget gene.

As used herein the term “isolated” refers to separated from its naturalenvironment. In the case of a dsRNA molecule, separated from thecytoplasm or the nucleus, conversely, in the case of a plant part suchas a seed, separated from the rest of the plant.

As used herein the term “isolated dsRNA molecule” refers to an isolatedRNA molecule which is substantially in a double stranded form. As usedherein, an isolated dsRNA molecule may be in solution and may includebuffers. An isolated dsRNA molecule is substantially separated fromother nucleic acid molecules including DNA.

As used herein the term “dsRNA” refers to two strands of anti-parallelpolyribonucleic acids held together by base pairing (e.g., two sequencesthat are the reverse complement of each other in the region of basepairing). The two strands can be of identical length or of differentlengths provided there is enough sequence homology between the twostrands that a double stranded structure is formed with at least 80%,90%, 95% or 100% complementarity over the entire length. As used herein,the term “overhang” refers to non-double stranded regions of a dsRNAmolecule (i.e., single stranded RNA). According to an embodiment of thedisclosure, there are no overhangs for the dsRNA molecule. According toanother embodiment of the disclosure, the dsRNA molecule comprises oneoverhang. According to other embodiments, a dsRNA molecule may comprisetwo overhangs.

In embodiments according to the present disclosure, an isolated dsRNAmolecule comprises a second strand having an RNA sequence that is atleast 80%, 90%, 95% or 100% complementary over its entire length to anantisense RNA sequence. In some embodiments, an isolated dsRNA moleculecomprises a second strand that is 99% complementary over its entirelength to an antisense RNA sequence. In other embodiments, the doublestranded region is 98% complementary over the entire length of anantisense RNA sequence. In yet other embodiments, the double strandedregion is 97% complementary over the entire length of an antisense RNAsequence. In further embodiments, the double stranded region maycomprise 96% of the entire length of an antisense RNA sequences. Incertain embodiments the double stranded region is between 90 and 100%complementary over the entire length of antisense RNA sequence. Incertain embodiments the double stranded region is between 95 and 100%complementary over the entire length of antisense RNA sequence.

The present disclosure provides for, and includes, embodiments of anisolated dsRNA molecule comprising a second strand having an RNAsequence that is nearly 100% complementary over its entire length to anantisense RNA sequence but having 1 mismatch. In some embodiments, thenearly 100% complementary dsRNA region may have 2 mismatches. In someembodiments, the nearly 100% complementary dsRNA region may have 3mismatches. Some embodiments according to the present disclosure providefor 4, 5 or 6 mismatches in a dsRNA region. In some embodiments, thenearly 100% complementary dsRNA region may have 1 or more, 2 or more, or3 or more mismatches.

According to an embodiment, an overhang may be 5′ to a double strandedregion comprising at least one antisense RNA sequence and its reversecomplement (e.g., 5′ to said antisense RNA sequence). According to anembodiment, an overhang may be 3′ to a double stranded region comprisingat least one antisense RNA sequence and its reverse complement (e.g., 3′to said antisense RNA sequence). In other embodiments according to thepresent disclosure, a dsRNA molecule may comprise two overhang regionsflanking a double stranded region.

According to other embodiments, an overhang region comprises less than10 bases. In certain embodiments, the strands are aligned such thatthere are at least 1, 2, or 3 bases at the end of the strands which donot align (i.e., for which no complementary bases occur in the opposingstrand) such that an overhang of 1, 2 or 3 residues occurs at one orboth ends of the duplex when strands are annealed. In an embodiment, aless than 10 base overhang may be a 5′ overhang (relative to the 5′ and3′ positions on the end of a double stranded RNA region). In anotherembodiment, a less than 10 base overhang may be a 3′ overhang. Relativeto a dsRNA molecule having at least one antisense RNA sequence, the 5′overhang may be located at 5′ of said antisense RNA sequences. In otherembodiments, the 5′ overhang may be located 3′ of said antisense RNAsequence (e.g., the 5′ overhang is on the complementary strand). Alsoprovided by the present disclosure are embodiments wherein the 3′overhang is located 3′ of said antisense RNA sequence or wherein the 3′overhang is located 5′ of said antisense RNA sequence. According toembodiments of the present disclosure, a 5′ overhanging sequence may be9 bases. In an embodiment, a 3′ overhanging sequence may be 9 bases.According to embodiments of the present disclosure, a 5′ overhangingsequence may be 8 bases. In an embodiment, a 3′ overhanging sequence maybe 8 bases. According to embodiments of the present disclosure, a 5′overhanging sequence may be 7 bases. In an embodiment, a 3′ overhangingsequence may be 7 bases. According to embodiments of the presentdisclosure, a 5′ overhanging sequence may be 6 bases. In an embodiment,a 3′ overhanging sequence may be 6 bases. In some embodiments, a singlestranded overhanging sequence may be less than 5 bases. According toembodiments of the present disclosure, a 5′ overhanging sequence may be5 bases. In an embodiment, a 3′ overhanging sequence may be 5 bases.According to embodiments of the present disclosure, a 5′ overhangingsequence may be 4 bases. In an embodiment, a 3′ overhanging sequence maybe 4 bases. According to embodiments of the present disclosure, a 5′overhanging sequence may be 3 bases. In an embodiment, a 3′ overhangingsequence may be 3 bases. According to embodiments of the presentdisclosure, a 5′ overhanging sequence may be 2 bases. In an embodiment,a 3′ overhanging sequence may be 2 bases.

As will be appreciated by one of ordinary skill in the art, a dsRNAmolecule of the present disclosure may refer to either strand of theanti-parallel nucleic acids. As will also be appreciated by one ofordinary skill in the art, a dsRNA molecule of the present disclosureincludes both a ‘sense’ and ‘antisense’ strand and that the sense andantisense strands are reverse complements of each other in a region ofbase pairing. As used herein the sequence of a dsRNA molecule forregulating a target gene of interest is provided as the ‘antisense’orientation with respect to the target gene of interest. Thus, one ofordinary skill in the art would appreciate that the 5′ end of a dsRNAmolecule for regulating a target gene of interest corresponds tosequences towards the 3′ end of the target gene of interest. Similarly,the 3′ end of a dsRNA molecule for regulating a target gene of interestcorresponds to sequences towards the 5′ end of a target gene ofinterest. As used herein, “the reverse complement of a dsRNA moleculefor regulating a target gene of interest” refers to a nucleic acidsequence in the ‘sense’ orientation.

The term “corresponding to the target gene of interest” or “dsRNA forregulating a target gene of interest” means that the dsRNA sequencecontains an RNA silencing agent to the target gene.

As used herein, the term “RNA silencing agent” refers to a nucleic acidwhich is capable of inhibiting or “silencing” the expression of a targetgene. In certain aspects, the RNA silencing agent is capable ofpreventing complete processing (e.g., the full translation and/orexpression) of an mRNA molecule through a post-transcriptional silencingmechanism. RNA silencing agents can be single- or double-stranded RNA orsingle- or double-stranded DNA or double-stranded DNA/RNA hybrids ormodified analogues thereof. In some aspects, the RNA silencing agentsare selected from the group consisting of (a) a single-stranded RNAmolecule (ssRNA), (b) a ssRNA molecule that self-hybridizes to form adouble-stranded RNA molecule, (c) a double-stranded RNA molecule(dsRNA), (d) a single-stranded DNA molecule (ssDNA), (e) a ssDNAmolecule that self-hybridizes to form a double-stranded DNA molecule,and (f) a single-stranded DNA molecule including a modified Pol III genethat is transcribed to an RNA molecule, (g) a double-stranded DNAmolecule (dsDNA), (h) a double-stranded DNA molecule including amodified Pol III promoter that is transcribed to an RNA molecule, (i) adouble-stranded, hybridized RNA/DNA molecule, or combinations thereof.In some aspects these polynucleotides include chemically modifiednucleotides or non-canonical nucleotides. In some aspects, the RNAsilencing agents are noncoding RNA molecules, for example RNA duplexescomprising paired strands, as well as precursor RNAs from which suchsmall non-coding RNAs can be generated. In some aspects, the RNAsilencing agents are dsRNAs such as siRNAs, miRNAs and shRNAs. In oneaspect, the RNA silencing agent is capable of inducing RNA interference.In another aspect, the RNA silencing agent is capable of mediatingtranslational repression. As used herein, an RNA silencing agent is atype of agent for regulating a target gene.

In some embodiments, the dsRNA molecule is subject to amplification byRNA-Dependent RNA Polymerase (RDRP). According to some embodiments, adsRNA molecule comprises a first strand having at least one antisenseRNA sequence for regulating a target gene, one or two heterologous RNAsequences corresponding to a smRNA, a helicase binding site and asequence encoding an RDRP, and a second complementary strand. Accordingto some embodiments, a dsRNA molecule comprises a first strand having atleast one antisense RNA sequence for regulating a target gene, one ortwo heterologous RNA sequences corresponding to a smRNA, a helicasebinding site and a sequence encoding an RDRP and further includingflanking 3′ UTR and 5′ UTR sequences from an endovirus and a second RNAstrand that is the reverse complement.

As used herein, “small RNA” or “smRNA” refers to RNA molecules thatfunction to modulate (e.g., inhibit), gene expression, and are presentin diverse eukaryotic organisms, including plants. As known to those ofskill in the art, smRNAs may be defined as low-molecular weight RNAsassociated with gene silencing and in some embodiments may be furtherdescribed as short (generally 21 to 26 nucleotides). Small RNAs includesiRNAs and miRNAs, which function in RNA silencing, also sometimesreferred to as RNA interference (RNAi). RNA silencing encompasses abroad range of phenomena in which large, double-stranded RNA, fold-backstructures, or stem-loop precursors are processed to about 21-26nucleotide (nt) small RNAs (e.g., siRNAs or miRNAs, which are describedfurther below) that then guide the cleavage of cognate RNAs, blockproductive translation thereof, or induce methylation of specific targetDNAs (Meins, F., et al., Annu Rev. Cell Dev. Biol., 21:297-318, 2005).

As used herein, a small RNA is an RNA molecule that is at least 15 basepairs in length, generally 15-30 nucleotides long, preferably 20-24nucleotides long. In some aspects, In aspects according to the presentdisclosure, a “small RNA” is greater than 30 base pairs in length. In anaspect, the small RNA is greater than 30 base pairs in length but lessthan about 600 base pairs. In an aspect, the small RNA is greater than100 base pairs in length but less than about 600 base pairs. In anaspect, the small RNA is greater than 200 base pairs in length but lessthan about 600 base pairs. A small RNA can be either double-stranded orsingle-stranded. Small RNA includes, without limitation, miRNA(microRNA), ta-siRNA (trans activating siRNA), siRNA, activating RNA(RNAa), nat-siRNA (natural anti-sense siRNA), he-siRNA (heterochromaticsiRNA), cis-acting siRNA, lmiRNA (long miRNA), lsiRNA (long siRNA) andeasiRNA (epigenetically activated siRNA) and their respectiveprecursors. Preferred siRNA molecules of the disclosure are miRNAmolecules, to-siRNA molecules and RNAa molecules and their respectiveprecursors. A small RNA may be processed in vivo by an organism to anactive form. According to aspects of the present disclosure, a selectiveinsecticide may be a small RNA. In embodiments according to the presentdisclosure a small RNA is a dsRNA.

As provided for and included in the present disclosure, a dsRNA moleculemay comprise an antisense RNA sequence for regulating a target gene ofinterest. In some embodiments, a dsRNA molecule for regulating a targetgene of interest may comprise an antisense RNA sequence that is greaterthan 30 base pairs in length to allow processing of the dsRNA in a plantcell and generation of secondary siRNA molecules. In other embodiments,a dsRNA molecule for regulating a target gene of interest may comprisean antisense RNA sequence that is from 30 to 600 bp in length to allowprocessing of the dsRNA in a plant cell and generation of secondarysiRNA molecules. As used herein, “secondary siRNA”, “phase RNA” and“ta-siRNA” or refer to dsRNA molecules generated after processing adsRNA molecule. In certain embodiments, the target gene regulation issilencing. In other embodiments, expression of the target gene isenhanced.

The present disclosure also includes and provides for embodiments havingdsRNA molecules having various lengths of dsRNA sequences, whereby theshorter version i.e., x is shorter or equals 50 bp (e.g., 17-50), isreferred to as siRNA or miRNA sequences. Longer dsRNA sequences of51-600 nucleotides are referred to herein as dsRNA, which can be furtherprocessed for siRNA molecules.

The term “siRNA” generally refers to small inhibitory RNA duplexes(generally between 17-30 base pairs, but also longer e.g., 31-50 bp)that induce the RNA interference (RNAi) pathway. In certain embodiments,siRNAs are chemically synthesized as 2 lmers with a central 19 bp duplexregion and symmetric 2-base 3′-overhangs on the termini, although it hasbeen recently described that chemically synthesized RNA duplexes of25-30 base length can have as much as a 100-fold increase in potencycompared with 21mers at the same location. Without being limited by anytheory, a role of siRNA is its involvement in the RNA interference(RNAi) pathway, where it interferes with the expression of a specificgene. Though not to be limiting, the observed increased potency obtainedusing longer RNAs in triggering RNAi is theorized to result fromproviding Dicer with a substrate (27mer) instead of a product (21mer)and that this improves the rate or efficiency of entry of the siRNAduplex into the RNA-induced silencing complex (RISC).

It has been found that position of the 3′-overhang influences potency ofan siRNA and asymmetric duplexes having a 3′-overhang on the antisensestrand are generally more potent than those with the 3′-overhang on thesense strand (Rose et al., 2005). This can be attributed to asymmetricalstrand loading into RISC, as the opposite efficacy patterns are observedwhen targeting the antisense transcript.

In some embodiments, the strands of a double-stranded interfering RNA(e.g., an siRNA) may be connected to form a hairpin or stem-loopstructure (e.g., an shRNA). Thus, as mentioned the RNA silencing agentof some embodiments of the disclosure may also be a short hairpin RNA(shRNA).

The term “shRNA”, as used herein, refers to an RNA agent having astem-loop structure, comprising a first and second region ofcomplementary sequence, the degree of complementarity and orientation ofthe regions being sufficient such that base pairing occurs between theregions, the first and second regions being joined by a loop region, theloop resulting from a lack of base pairing between nucleotides (ornucleotide analogs) within the loop region. The number of nucleotides inthe loop is a number between and including 3 to 23, 5 to 15, 7 to 13, 4to 9, or 9 to 11. Some of the nucleotides in the loop can be involved inbase-pair interactions with other nucleotides in the loop. Examples ofoligonucleotide sequences that can be used to form the loop include5′-UUCAAGAGA-3′ (Brummelkamp, T. R. et al. (2002) Science 296: 550) and5′-UUUGUGUAG-3′ (Castanotto, D. et al. (2002) RNA 8:1454). It will berecognized by one of skill in the art that the resulting single chainoligonucleotide forms a stem-loop or hairpin structure comprising adouble-stranded region capable of interacting with the RNAi machinery.

As used herein, the phrase “microRNA (also referred to hereininterchangeably as “miRNA” or “miR”) or a precursor thereof” refers to amicroRNA (miRNA) molecule acting as a post-transcriptional regulator.Typically, the miRNA molecules are RNA molecules of about 20 to 22nucleotides in length which can be loaded into a RISC complex and whichdirect the cleavage of another RNA molecule, wherein the other RNAmolecule comprises a nucleotide sequence essentially complementary tothe nucleotide sequence of the miRNA molecule.

While not limited by a particular theory, a miRNA molecule is oftenprocessed from a “pre-miRNA” or as used herein a precursor of a miRNAmolecule by proteins, such as DCL proteins. Pre-microRNA molecules aretypically processed from pri-microRNA molecules (primary transcripts).The single-stranded RNA segments flanking the pre-microRNA are importantfor processing of the pri-miRNA into the pre-miRNA. The cleavage siteappears to be determined by the distance from the stem-ssRNA junction(Han et al., 2006, Cell, 125:887-901). In some embodiments, a miRNAmolecule is loaded onto a RISC complex where it can guide the cleavageof the target gene of interest.

Pre-microRNA molecules are typically processed from pri-microRNAmolecules (primary transcripts). The single stranded RNA segmentsflanking the pre-microRNA are important for processing of the pri-miRNAinto the pre-miRNA. The cleavage site appears to be determined by thedistance from the stem-ssRNA junction (Han et al. 2006, Cell 125,887-901, 887-901).

As used herein, a “pre-miRNA” molecule is an RNA molecule of about 100to about 200 nucleotides, preferably about 100 to about 130 nucleotideswhich can adopt a secondary structure comprising an imperfect doublestranded RNA stem and a single stranded RNA loop (also referred to as“hairpin”) and further comprising the nucleotide sequence of the miRNA(and its complement sequence) in the double stranded RNA stem. Accordingto a specific embodiment, the miRNA and its complement are located about10 to about 20 nucleotides from the free ends of the miRNA doublestranded RNA stem. The length and sequence of the single stranded loopregion are not critical and may vary considerably, e.g. between 30 and50 nucleotides in length. The complementarity between the miRNA and itscomplement need not be perfect and about 1 to 3 bulges of unpairednucleotides can be tolerated. The secondary structure adopted by an RNAmolecule can be predicted by computer algorithms conventional in the artsuch as mFOLD. The particular strand of the double stranded RNA stemfrom the pre-miRNA which is released by DCL activity and loaded onto theRISC complex is determined by the degree of complementarity at the 5′end, whereby the strand which at its 5′ end is the least involved inhydrogen bounding between the nucleotides of the different strands ofthe cleaved dsRNA stem is loaded onto the RISC complex and willdetermine the sequence specificity of the target RNA moleculedegradation. However, if empirically the miRNA molecule from aparticular synthetic pre-miRNA molecule is not functional (because the“wrong” strand is loaded on the RISC complex), it will be immediatelyevident that this problem can be solved by exchanging the position ofthe miRNA molecule and its complement on the respective strands of thedsRNA stem of the pre-miRNA molecule. As is known in the art, bindingbetween A and U involving two hydrogen bounds, or G and U involving twohydrogen bounds is less strong that between G and C involving threehydrogen bounds.

In some embodiments according to the present disclosure, naturallyoccurring miRNA molecules may be comprised within their naturallyoccurring pre-miRNA molecules. In other embodiments, a miRNA can beintroduced into a non-natural heterologous pre-miRNA molecule scaffoldby exchanging the nucleotide sequence of the miRNA molecule. Thus, whenprocessed the recombinant pre-miRNA produces an miRNA having a replacedsequence. In some embodiments, the scaffold of the pre-miRNA can also becompletely synthetic. Likewise, synthetic miRNA molecules may becomprised within, and processed from, existing pre-miRNA moleculescaffolds or synthetic pre-miRNA scaffolds. Some pre-miRNA scaffolds maybe preferred over others for their efficiency to be correctly processedinto the designed microRNAs, particularly when expressed as a chimericgene. In some aspects a chimeric pre-miRNA gene may include other DNAregions, such as untranslated leader sequences, transcriptiontermination and polyadenylation regions that are incorporated in theprimary transcript in addition to the pre-microRNA.

According to the present teachings, the dsRNA sequences may be naturallyoccurring or synthetic.

The dsRNA sequence for regulating a target gene of interest may containmultiple discrete portions or regions that correspond to the targetgene, separated by portions that do not correspond to the target gene.In some embodiments, portions that do not correspond to the target genemay optionally correspond to a second, third, or fourth target gene. Itwill be appreciated that the portions that correspond to the target genemay have different lengths, different degrees of sequence identity tothe target gene, and may correspond to regions located anywhere withinthe target gene.

In embodiments according to the present disclosure, an antisense RNAsequence may be flanked by two nucleic acid sequences that arecomplementary to an smRNA expressed in the plant. In some embodiments,the two flanking nucleic acid sequences may be complementary to twodifferent smRNAs. In yet other embodiments, the two flanking nucleicacid sequences may comprise sequences that are complementary to morethan two smRNAs. In further embodiments, the smRNAs may comprise twocopies of one smRNA and a nucleic acid sequence complementary to adifferent smRNA.

The present disclosure provides for an includes dsRNA moleculescomprising an antisense RNA sequence and one or two nucleic acidsequences that are complementary to an smRNA expressed in the plant. Thepresent disclosure provides for embodiments having the composition andorientation of one or two nucleic acid sequences that are complementaryto an smRNA expressed in the plant of dsRNA molecules as shown in Table1 below. In certain embodiments, the dsRNA molecules of Table 1 furthercomprise a helicase binding sequence. In some embodiments, the dsRNAmolecules of Table 1, further comprise a helicase binding sequence and aRDRP polypeptide encoding sequence.

TABLE 1 Embodiments of first strands of dsRNA molecules having a firstand second nucleic acid sequence complementary to an smRNA expressed ina plant 5′ 5′ Antisense RNA 3′ 3′ Construct smRNA₂ smRNA₁ sequencesmRNA₁ smRNA₂ A01¹ None None Present None None A02 None Direct PresentDirect None A03 None Direct Present Direct Mut None A04 None DirectPresent R/C None A05 None Direct Present R/C Mut None A06 None DirectMut Present Direct None A07 None Direct Mut Present Direct Mut None A08None Direct Mut Present R/C None A09 None Direct Mut Present R/C MutNone A10 None R/C Present Direct None A11 None R/C Present Direct MutNone A12 None R/C Present R/C None A13 None R/C Present R/C Mut None A14None R/C Mut Present Direct None A15 None R/C Mut Present Direct MutNone A16 None R/C Mut Present R/C None A17 None R/C Mut Present R/C MutNone B01 Direct Direct Present Direct Direct B02 Direct Direct PresentDirect Direct Mut B03 Direct Direct Present Direct R/C B04 Direct DirectPresent Direct R/C Mut B05 Direct Direct Present Direct Mut Direct B06Direct Direct Present Direct Mut Direct Mut B07 Direct Direct PresentDirect Mut R/C B08 Direct Direct Present Direct Mut R/C Mut B09 DirectDirect Present R/C Direct B10 Direct Direct Present R/C Direct Mut B11Direct Direct Present R/C R/C B12 Direct Direct Present R/C R/C Mut B13Direct Direct Present R/C Mut Direct B14 Direct Direct Present R/C MutDirect Mut B15 Direct Direct Present R/C Mut R/C B16 Direct DirectPresent R/C Mut R/C Mut B17 Direct Direct Mut Present Direct Direct B18Direct Direct Mut Present Direct Direct Mut B19 Direct Direct MutPresent Direct R/C B20 Direct Direct Mut Present Direct R/C Mut B21Direct Direct Mut Present Direct Mut Direct B22 Direct Direct MutPresent Direct Mut Direct Mut B23 Direct Direct Mut Present Direct MutR/C B24 Direct Direct Mut Present Direct Mut R/C Mut B25 Direct DirectMut Present R/C Direct B26 Direct Direct Mut Present R/C Direct Mut B27Direct Direct Mut Present R/C R/C B28 Direct Direct Mut Present R/C R/CMut B29 Direct Direct Mut Present R/C Mut Direct B30 Direct Direct MutPresent R/C Mut Direct Mut B31 Direct Direct Mut Present R/C Mut R/C B32Direct Direct Mut Present R/C Mut R/C Mut B33 Direct R/C Present DirectDirect B34 Direct R/C Present Direct Direct Mut B35 Direct R/C PresentDirect R/C B36 Direct R/C Present Direct R/C Mut B37 Direct R/C PresentR/C Direct B38 Direct R/C Present R/C Direct Mut B39 Direct R/C PresentR/C R/C B40 Direct R/C Present R/C R/C Mut B41 Direct R/C Present R/CMut Direct B42 Direct R/C Present R/C Mut Direct Mut B43 Direct R/CPresent R/C Mut R/C B44 Direct R/C Present R/C Mut R/C Mut B45 DirectR/C Mut Present Direct Direct B46 Direct R/C Mut Present Direct DirectMut B47 Direct R/C Mut Present Direct R/C B48 Direct R/C Mut PresentDirect R/C Mut B49 Direct R/C Mut Present Direct Mut Direct B50 DirectR/C Mut Present Direct Mut Direct Mut B51 Direct R/C Mut Present DirectMut R/C B52 Direct R/C Mut Present Direct Mut R/C Mut B53 Direct R/C MutPresent R/C Direct B54 Direct R/C Mut Present R/C Direct Mut B55 DirectR/C Mut Present R/C R/C B56 Direct R/C Mut Present R/C R/C Mut B57Direct R/C Mut Present R/C Mut Direct B58 Direct R/C Mut Present R/C MutDirect Mut B59 Direct R/C Mut Present R/C Mut R/C B60 Direct R/C MutPresent R/C Mut R/C Mut C01 Direct Mut Direct Present Direct Direct C02Direct Mut Direct Present Direct Direct Mut C03 Direct Mut DirectPresent Direct R/C C04 Direct Mut Direct Present Direct R/C Mut C05Direct Mut Direct Present Direct Mut Direct C06 Direct Mut DirectPresent Direct Mut Direct Mut C07 Direct Mut Direct Present Direct MutR/C C08 Direct Mut Direct Present Direct Mut R/C Mut C09 Direct MutDirect Present R/C Direct C10 Direct Mut Direct Present R/C Direct MutC11 Direct Mut Direct Present R/C R/C C12 Direct Mut Direct Present R/CR/C Mut C13 Direct Mut Direct Present R/C Mut Direct C14 Direct MutDirect Present R/C Mut Direct Mut C15 Direct Mut Direct Present R/C MutR/C C16 Direct Mut Direct Present R/C Mut R/C Mut C17 Direct Mut DirectMut Present Direct Direct C18 Direct Mut Direct Mut Present DirectDirect Mut C19 Direct Mut Direct Mut Present Direct R/C C20 Direct MutDirect Mut Present Direct R/C Mut C21 Direct Mut Direct Mut PresentDirect Mut Direct C22 Direct Mut Direct Mut Present Direct Mut DirectMut C23 Direct Mut Direct Mut Present Direct Mut R/C C24 Direct MutDirect Mut Present Direct Mut R/C Mut C25 Direct Mut Direct Mut PresentR/C Direct C26 Direct Mut Direct Mut Present R/C Direct Mut C27 DirectMut Direct Mut Present R/C R/C C28 Direct Mut Direct Mut Present R/C R/CMut C29 Direct Mut Direct Mut Present R/C Mut Direct C30 Direct MutDirect Mut Present R/C Mut Direct Mut C31 Direct Mut Direct Mut PresentR/C Mut R/C C32 Direct Mut Direct Mut Present R/C Mut R/C Mut C33 DirectMut R/C Present Direct Direct C34 Direct Mut R/C Present Direct DirectMut C35 Direct Mut R/C Present Direct R/C C36 Direct Mut R/C PresentDirect R/C Mut C37 Direct Mut R/C Present R/C Direct C38 Direct Mut R/CPresent R/C Direct Mut C39 Direct Mut R/C Present R/C R/C C40 Direct MutR/C Present R/C R/C Mut C41 Direct Mut R/C Present R/C Mut Direct C42Direct Mut R/C Present R/C Mut Direct Mut C43 Direct Mut R/C Present R/CMut R/C C44 Direct Mut R/C Present R/C Mut R/C Mut C45 Direct Mut R/CMut Present Direct Direct C46 Direct Mut R/C Mut Present Direct DirectMut C47 Direct Mut R/C Mut Present Direct R/C C48 Direct Mut R/C MutPresent Direct R/C Mut C49 Direct Mut R/C Mut Present Direct Mut DirectC50 Direct Mut R/C Mut Present Direct Mut Direct Mut C51 Direct Mut R/CMut Present Direct Mut R/C C52 Direct Mut R/C Mut Present Direct Mut R/CMut C53 Direct Mut R/C Mut Present R/C Direct C54 Direct Mut R/C MutPresent R/C Direct Mut C55 Direct Mut R/C Mut Present R/C R/C C56 DirectMut R/C Mut Present R/C R/C Mut C57 Direct Mut R/C Mut Present R/C MutDirect C58 Direct Mut R/C Mut Present R/C Mut Direct Mut C59 Direct MutR/C Mut Present R/C Mut R/C C60 Direct Mut R/C Mut Present R/C Mut R/CMut D01 R/C Direct Present Direct Direct D02 R/C Direct Present DirectDirect Mut D03 R/C Direct Present Direct R/C D04 R/C Direct PresentDirect R/C Mut D05 R/C Direct Present Direct Mut Direct D07 R/C DirectPresent Direct Mut R/C D08 R/C Direct Present Direct Mut R/C Mut D10 R/CDirect Present R/C Direct Mut D11 R/C Direct Present R/C R/C D12 R/CDirect Present R/C R/C Mut D13 R/C Direct Present R/C Mut Direct D14 R/CDirect Present R/C Mut Direct Mut D15 R/C Direct Present R/C Mut R/C D16R/C Direct Present R/C Mut R/C Mut D17 R/C Direct Mut Present DirectDirect D18 R/C Direct Mut Present Direct Direct Mut D19 R/C Direct MutPresent Direct R/C D20 R/C Direct Mut Present Direct R/C Mut D21 R/CDirect Mut Present Direct Mut Direct D22 R/C Direct Mut Present DirectMut Direct Mut D23 R/C Direct Mut Present Direct Mut R/C D24 R/C DirectMut Present Direct Mut R/C Mut D25 R/C Direct Mut Present R/C Direct D26R/C Direct Mut Present R/C Direct Mut D27 R/C Direct Mut Present R/C R/CD28 R/C Direct Mut Present R/C R/C Mut D29³ R/C Direct Mut Present R/CMut Direct D30 R/C Direct Mut Present R/C Mut Direct Mut D31 R/C DirectMut Present R/C Mut R/C D32 R/C Direct Mut Present R/C Mut R/C Mut D33R/C R/C Present Direct Direct D34 R/C R/C Present Direct Direct Mut D35R/C R/C Present Direct R/C D36 R/C R/C Present Direct R/C Mut D37 R/CR/C Present R/C Direct D38 R/C R/C Present R/C Direct Mut D39 R/C R/CPresent R/C R/C D40 R/C R/C Present R/C R/C Mut D41 R/C R/C Present R/CMut Direct D42 R/C R/C Present R/C Mut Direct Mut D43 R/C R/C PresentR/C Mut R/C D44 R/C R/C Present R/C Mut R/C Mut D45 R/C R/C Mut PresentDirect Direct D46 R/C R/C Mut Present Direct Direct Mut D47 R/C R/C MutPresent Direct R/C D48 R/C R/C Mut Present Direct R/C Mut D49 R/C R/CMut Present Direct Mut Direct D50 R/C R/C Mut Present Direct Mut DirectMut D51 R/C R/C Mut Present Direct Mut R/C D52 R/C R/C Mut PresentDirect Mut R/C Mut D53 R/C R/C Mut Present R/C Direct D54 R/C R/C MutPresent R/C Direct Mut D55 R/C R/C Mut Present R/C R/C D56 R/C R/C MutPresent R/C R/C Mut D57 R/C R/C Mut Present R/C Mut Direct D58 R/C R/CMut Present R/C Mut Direct Mut D59 R/C R/C Mut Present R/C Mut R/C D6R/C Direct Present Direct Mut Direct Mut D60 R/C R/C Mut Present R/C MutR/C Mut D9 R/C Direct Present R/C Direct E01 R/C Mut Direct PresentDirect Direct E02 R/C Mut Direct Present Direct Direct Mut E03 R/C MutDirect Present Direct R/C E04 R/C Mut Direct Present Direct R/C Mut E05R/C Mut Direct Present Direct Mut Direct E06 R/C Mut Direct PresentDirect Mut Direct Mut E07 R/C Mut Direct Present Direct Mut R/C E08 R/CMut Direct Present Direct Mut R/C Mut E09 R/C Mut Direct Present R/CDirect E10 R/C Mut Direct Present R/C Direct Mut E11 R/C Mut DirectPresent R/C R/C E12 R/C Mut Direct Present R/C R/C Mut E13 R/C MutDirect Present R/C Mut Direct E14 R/C Mut Direct Present R/C Mut DirectMut E15 R/C Mut Direct Present R/C Mut R/C E16 R/C Mut Direct PresentR/C Mut R/C Mut E17 R/C Mut Direct Mut Present Direct Direct E18 R/C MutDirect Mut Present Direct Direct Mut E19 R/C Mut Direct Mut PresentDirect R/C E20 R/C Mut Direct Mut Present Direct R/C Mut E21 R/C MutDirect Mut Present Direct Mut Direct E22 R/C Mut Direct Mut PresentDirect Mut Direct Mut E23 R/C Mut Direct Mut Present Direct Mut R/C E24R/C Mut Direct Mut Present Direct Mut R/C Mut E25 R/C Mut Direct MutPresent R/C Direct E26 R/C Mut Direct Mut Present R/C Direct Mut E27 R/CMut Direct Mut Present R/C R/C E28 R/C Mut Direct Mut Present R/C R/CMut E29 R/C Mut Direct Mut Present R/C Mut Direct E30 R/C Mut Direct MutPresent R/C Mut Direct Mut E31 R/C Mut Direct Mut Present R/C Mut R/CE32 R/C Mut Direct Mut Present R/C Mut R/C Mut E33 R/C Mut R/C PresentDirect Direct E34 R/C Mut R/C Present Direct Direct Mut E35 R/C Mut R/CPresent Direct R/C E36 R/C Mut R/C Present Direct R/C Mut E37 R/C MutR/C Present R/C Direct E38 R/C Mut R/C Present R/C Direct Mut E39 R/CMut R/C Present R/C R/C E40 R/C Mut R/C Present R/C R/C Mut E41 R/C MutR/C Present R/C Mut Direct E42 R/C Mut R/C Present R/C Mut Direct MutE43 R/C Mut R/C Present R/C Mut R/C E44 R/C Mut R/C Present R/C Mut R/CMut E45 R/C Mut R/C Mut Present Direct Direct E46 R/C Mut R/C MutPresent Direct Direct Mut E47 R/C Mut R/C Mut Present Direct R/C E48 R/CMut R/C Mut Present Direct R/C Mut E49 R/C Mut R/C Mut Present DirectMut Direct E50 R/C Mut R/C Mut Present Direct Mut Direct Mut E51 R/C MutR/C Mut Present Direct Mut R/C E52 R/C Mut R/C Mut Present Direct MutR/C Mut E53 R/C Mut R/C Mut Present R/C Direct E54 R/C Mut R/C MutPresent R/C Direct Mut E55 R/C Mut R/C Mut Present R/C R/C E56 R/C MutR/C Mut Present R/C R/C Mut E57 R/C Mut R/C Mut Present R/C Mut DirectE58 R/C Mut R/C Mut Present R/C Mut Direct Mut E59 R/C Mut R/C MutPresent R/C Mut R/C E60 R/C Mut R/C Mut Present R/C Mut R/C Mut ¹seeconstruct #1, FIG. 2B and construct #8, FIG. 8; 2: See construct #2,FIG. 2A and 2C; construct #4 FIG. 4; construct #9 FIG. 9 ³see construct,#3 and construct #6 As used herein, “Direct” means the direct sequence(i.e., a sequence having the same order of nucleotides and in the sameorientation) of a smRNA; “Direct Mut” means the direct sequence of ansmRNA having a mutation that renders it resistant to cleavage; “R/C”means the reverse complement of an smRNA; and “R/C Mut” means a reversecomplement of an smRNA having a mutation that renders it resistant tocleavage.

In embodiments according to the present disclosure, the sequences of theembodiments of Table 1 include, but are not limited to combinations ofSEQ ID NOs: 26 to 35, and 41 to 288, their complements, andnon-cleavable mutants thereof. In some embodiments, RNA sequence forregulating a target gene of interest comprises a nucleic acid having 90to 100% homology to a sequence selected from the group consisting of SEQID NOs:8, 11, 12, 36 to 38, and their complements thereof. It isunderstood that the present disclosure provides for, and includes, dsRNAconstructs of Table 1 having a second reverse complementary strand atleast to the antisense RNA sequence. The present disclosure furtherprovides dsRNA constructs having a second reverse complimentary strandcomprising an antisense RNA sequence and a 5′ smRNA₁ sequence. Thepresent disclosure further provides dsRNA constructs having a secondreverse complimentary strand comprising an antisense RNA sequence, a 5′smRNA₁ sequence and a 3′ smRNA₁ sequence. The present disclosure furtherprovides dsRNA constructs having a second reverse complimentary strandcomprising an antisense RNA sequence, a 5′ smRNA₁ sequence, a 5′ smRNA₂sequence and a 3′ smRNA₁ sequence. The present disclosure furtherprovides dsRNA constructs having a second reverse complimentary strandcomprising an antisense RNA sequence, a 5′ smRNA₁ sequence, a 5′ smRNA₂sequence, a 3′ smRNA₁ sequence and a 4′ smRNA₂ sequence.

The present disclosure provides for and includes second reversecomplementary strand of the constructs of Table 1 having mismatches. Insome embodiments, the second reverse complementary strand provides for adouble stranded region comprising a smRNA and its non-cleavable mutant.Accordingly it is understood, the dsRNA comprises one or more mismatchescorresponding to the mismatch between the smRNA and its non-cleavablemutant. The present disclosure provides for combinations of the firststrands of Table 1 to produce dsRNA molecules.

Accordingly, it is understood that construct A02 may be combined with,for example, the reverse complement of A02 to prepare a dsRNA of thepresent disclosure. In other embodiments, for example, construct A02 maybe combined with the reverse complement of construct A06 to prepare adsRNA of the present disclosure having a mismatch sequence at thenon-cleavable site. One of ordinary skill in the art would recognizethat additional combinations of the constructs of Table 1 may beprepared in accordance with the present disclosure.

In embodiments according to the present disclosure, the sequencecomplementarity may be, but are not required to be, 100%. In certainembodiments of the disclosure the degree of complementarity, e.g.,percent complementarity, need only be sufficient to provide for stablebinding of a smRNA to the complementary site. In certain embodiments ofthe disclosure the degree of complementarity need only be sufficientsuch that the smRNA pairs to the complementary site and mediatescleavage of the target mRNA. For example, in certain embodiments of thedisclosure the degree of complementarity is at least 70%, at least 80%,or at least 90%. In certain embodiments of the disclosure the number ofmismatched or unpaired nucleotides in the siRNA strand or miRNA,following binding to the complementary site, is between 0 and 5, e.g.,1, 2, 3, 4, or 5.

In other embodiments according to the present disclosure, the sequencecomplementarity of an smRNA may be greater than 90%. In someembodiments, the sequence complementarity of an smRNA may be greaterthan 91%. In some embodiments, the sequence complementarity of an smRNAmay be greater than 92%. In some embodiments, the sequencecomplementarity of an smRNA may be greater than 93%. In someembodiments, the sequence complementarity of an smRNA may be greaterthan 94%. In some embodiments, the sequence complementarity of an smRNAmay be greater than 95%. In some embodiments, the sequencecomplementarity of an smRNA may be greater than 96%. In someembodiments, the sequence complementarity of an smRNA may be greaterthan 97%. In some embodiments, the sequence complementarity of an smRNAmay be greater than 98%. In some embodiments, the sequencecomplementarity of an smRNA may be greater than 99%. In someembodiments, the sequence complementarity of an smRNA may be 100%. Inembodiments according to the present disclosure, sequencecomplementarity may be between 90 and 100% or 95 and 100%. According toembodiments of the present disclosure the smRNAs may be selected fromthe group consisting of SEQ ID NOs 26 to 35, 41 to 288, andnon-cleavable mutants thereof.

As used herein, the terms “complementarity” and “complementary” refer toa nucleic acid that can form one or more hydrogen bonds with anothernucleic acid sequence by either traditional Watson-Crick or othernon-traditional types of interactions. It will be recognized thatcomplementarity and homology or identity are related terms. That is, ahomology describes the degree of similarity between two or morenucleotide sequences when examined in the same 5′ to 3′ orientation. Incontrast, complementarity describes the degree of similarity between twoor more nucleotide sequences when comparing a sequence having a 5′ to 3′orientation to a sequence having a 3′ to 5′ orientation. Thus, a firstand second sequence having 90% homology will also have 90%complementarity when the first sequence is compared to the reversecomplement of the second sequences. In reference to the nucleicmolecules of the presently disclosed subject matter, the binding freeenergy of a nucleic acid molecule with its complementary sequence issufficient to allow the relevant function of the nucleic acid toproceed, in some embodiments, to form a duplex structure underphysiological conditions in a plant cell, to mediate ribonucleaseactivity, etc. For example, the degree of complementarity between thesense and antisense strands of an miRNA precursor can be the same ordifferent from the degree of complementarity between themiRNA-containing strand of an miRNA precursor and the target nucleicacid sequence. Determination of binding free energies for nucleic acidmolecules is well known in the art. See e.g., Freier et al., 1986;Turner et al., 1987. One of ordinary skill in the art would be able totest for sufficiency of complementarity by random or site directedmutagenesis and screening of silencing activity and dsRNA moleculestability in vivo.

In certain embodiments, the phrase “percent complementarity” refers tothe percentage of residues in a nucleic acid molecule that can formhydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleicacid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%,80%, 90%, and 100% complementary). The terms “100% complementary”,“fully complementary”, and “perfectly complementary” indicate that allof the contiguous residues of a nucleic acid sequence can hydrogen bondwith the same number of contiguous residues in a second nucleic acidsequence. It will be appreciated that the nucleic acids may havedifferent lengths and/or that there may be bulges when the two nucleicacids are optimally aligned for maximum complementarity over a givenportion of either sequence. Percent complementarity can, in variousembodiments of the disclosure, disregard such bulges in the computationor consider the percentage complementarity to be the number of paired(hydrogen bonded) residues divided by the total number of residues overa given length, which may be the length of the shorter or the longernucleic acid in different embodiments.

Any complementary sequence for a smRNA may be used in variousembodiments of the present disclosure. The sequence may be complementaryto an miRNA or siRNA. The sequence may be perfectly (100%) complementaryor may have imperfect complementarity as described herein and known inthe art. The complementary sequence may be one that is naturally foundin a trans-acting-siRNA-producing (TAS) locus or to-siRNA precursor RNA,flanking the portion of the RNA that is cleaved to produce ta-siRNAs.The complementary sequence may be any smRNA complementary sequence thatis found on one side of a nucleic acid sequence that is cleaved toproduce siRNA, wherein a second smRNA complementary sequence is found onthe other side of the nucleic acid sequence. In various embodiments ofthe disclosure the complementary sequence is recognized by a smRNAselected from the group consisting of:

miR390: (SEQ ID NO: 25) AAGCUCAGGAGGGAUAGCGCC; miR161.1: (SEQ ID NO: 26)UUGAAAGUGACUACAUCGGGG; miR400: (SEQ ID NO: 27) UAUGAGAGUAUUAUAAGUCAC;TAS2 3′D6(−): (SEQ ID NO: 28) AUAUCCCAUUUCUACCAUCUG; TAS 1b 3′D4(−):(SEQ ID NO: 29) UUCUUCUACCAUCCUAUCAAU; TAS3 5′D7(+): (SEQ ID NO: 30)UUCUUGACCUUGUAAGACCCC; TAS3 5′D8(+): (SEQ ID NO: 31)UUCUUGACCUUGUAAGGCCUU; miR168: (SEQ ID NO: 32) UCGCUUGGUGCAGGUCGGGAA;miR828 (SEQ ID NO: 33) UCUUGCUUAAAUGAGUAUUCCA; and miR393:(SEQ ID NO: 34) UCCAAAGGGAUCGCAUUGAUC.

In one embodiment, the miRNA is UUCGCUUGCAGAGAGAAAUCAC (SEQ ID NO: 35).Note that these sequences may have been identified in one or moreplants, e.g., Arabidopsis, most land plants, moss, etc. It will beappreciated that in some cases the sequences are conserved acrossmultiple species while in other cases there could be minor variations.Such variations are encompassed within the present disclosure. It willbe appreciated that homologous siRNAs or miRNAs from other plant speciesthan those listed could be used. Optionally, recognition of thecomplementary sequence by the cognate miRNA or siRNA leads to cleavage.One of skill in the art could determine whether binding and/or cleavageof a smRNA to a candidate complementary sequence occurs in vivo (inliving cells or organisms) or in vitro, e.g., under conditionsapproximating physiological intercellular conditions. In otherembodiments, an smRNA may be selected from the group consisting of SEQID NOs:41 to 288 (see, Table 1 of U.S. Pat. No. 8,143,480).

The length of the complementary sequence could vary. The length of acomplementary sequence may be defined as equal to the length of thesmRNA that binds to it, but it will be appreciated that a complementarysequence could differ in length from that of the smRNA, e.g., it may beshorter than the length of the smRNA. Typically the complementarysequence is sufficiently long such that the smRNA can bind (e.g.,hybridize) to the sequence with reasonable specificity and, optionally,direct cleavage within a duplex structure formed upon binding. Suchcleavage may occur at a position within the duplex typical of cleavagedirected by smRNAs, (e.g., in certain embodiments at position 10 or 11of the smRNA). For example, a complementary sequence could be between 15and 24 nucleotides in length, or any intervening number, wherein thereare 1, 2, 3, 4, or 5 mismatches when the smRNA is paired with thecomplementary sequence in the case of a 15 nucleotide sequence and up to6, 7, or 8 mismatches in the case of a 24 nucleotide complementarysequence. Similar considerations would apply for other smRNAcomplementary sequence. It will be appreciated that there may be“bulges” in the duplex formed when an smRNA pairs with its complementarysequence. In such instances a bulge could be considered equivalent to asingle mismatch or, in various embodiments of the disclosure a bulge ofX nucleotides could be considered equivalent to X mismatches. It willalso be appreciated that the specificity of binding of the smRNA to thecomplementary sequence need not be completely specific, e.g., the smRNAmay bind to different sequence having either a lesser or greater degreeof complementarity.

In embodiments according to the present disclosure, a first or secondcomplementary sequence may be between 15 and 30 nt, between 18 and 24nt, between 20 and 22, or exactly 21 nt in length. In some embodiments afirst or second complementary sequence may comprise any interveningrange or specific value within the foregoing ranges in certainembodiments of the disclosure. In certain non-limiting embodiments ofthe disclosure the number of mismatched or unpaired nucleotides in thesiRNA strand or miRNA, following binding to the complementary sequence,is between 0 and 5, e.g., 1, 2, 3, 4, or 5. In certain non-limitingembodiments of the disclosure the number of mismatched or unpairednucleotides (including those in both strands) in a duplex structureformed between the smRNA and its complementary sequence, is between 0and 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The mismatches or bulgesmay occur at any position within the duplex structure, in variousembodiments of the disclosure. In certain embodiments the mismatches orbulges are located at positions known in the art not to typicallyinhibit or prevent smRNA-directed cleavage. In other embodiments themismatches or bulges are located at such positions. One or moremismatches or bulges may occur, for example, at any position withrespect to the 5′ end of a smRNA depicted in the figures herein orcontemplated when an smRNA described herein pairs with a sequencecomplementary to it. The mismatch may be any mismatch known in the art.In certain embodiments a mismatch is said to occur when a nucleotidewithin an at least partly double-stranded structure is not paired in aconventional G-C, A-T, or A-U base pair. In certain embodiments amismatch is said to occur when a nucleotide in an at least partlydouble-stranded structure is not paired in a Watson-Crick base pair. Itwill be appreciated that the aforementioned mismatches may exclude“bulges”, wherein a nucleotide bulges outward from an otherwise duplexregion by being located between two nucleotides that are base pairedwith adjacent nucleotides on the opposite strand of the duplex.

The present disclosure further includes and provides for embodimentswherein a first and a second complementary sequence are the same length.In other embodiments, a first complementary sequence may be a differentlength than a second complementary sequence.

The portion of the smRNA that is complementary to the complementarysequence could vary. For example, in certain embodiments thecomplementary site is at least 70%, at least 80%, or at least 90%complementary to the first 16 nucleotides of the smRNA. In certainembodiments the complementary sequence is at least 70%, at least 80%, orat least 90% complementary to the first 17, 18, or 19 nucleotides of thesmRNA. In certain embodiments the complementary sequence is asubsequence of a complementary sequence, wherein said subsequence is atleast 16, 17, 18, 19, 20, or 21 nucleotides in length. In someembodiments the subsequence is the last 16, 17, 18, 19, 20, or 21nucleotides of the listed sequence. In some embodiments the subsequenceis at least 70%, at least 80%, at least 90%, or 100% complementary tothe first 16, 17, 18, 19, 20, or 21 nucleotides of an smRNA.

The present disclosure also includes, and provides for, dsRNA moleculeshaving a single smRNA complementary sequence and an antisense RNAsequence for regulating a target gene of interest. In embodimentsaccording to the present disclosure, where the dsRNA molecule ispresented as an antisense sequence, an smRNA sequence on the 5′ end isan upstream sequences. In other embodiments, an smRNA sequence on the 3′end is a downstream sequence. Not to be limited by theory, a smRNAcomplementary sequence binds (hybridizes) to a complementary smRNA andthe duplex recruits an RDRP, such as RNA-dependent RNA polymerase 6(RDR6). The dsRNA molecule can be further processed by Dicer-likeenzymes such as dicer-like protein 4 (DCL4) to produce the siRNAs. Itwill be appreciated that the presence of a second complementary sequence(e.g., flanking sequences) depends on the sequence of smRNA that thecomplementary sequence recognizes. In some instances (i.e., miR390) itis required whereas in others it is not required (i.e., miR173). In someaspects, for smRNAs that do not require a second complementary sequence(flanking sequence), the inclusion of a second complementary sequencemay increase the propensity, efficiency or rate of generating siRNAs.

When only one smRNA complementary sequence is present in a dsRNAmolecule, then it can be upstream (5′) or downstream (3′) to the dsRNAsequence for silencing the target gene (where the dsRNA sequence isreferenced in the antisense orientation). According to a specificembodiment, this smRNA complementary sequence is functional and islocated 3′ to the dsRNA sequence for silencing the target gene.According to another specific embodiment, this smRNA complementarysequence is functional (e.g., miR173) and is located 5′ to the dsRNAsequence for silencing the target gene.

When two complementary sequences to one or more an smRNAs are included,one sequence (e.g., the first site) is located 5′ to the dsRNA sequencefor silencing the target gene (where the dsRNA sequence is referenced inthe antisense orientation), and the second site is located 3′ to thedsRNA sequence for silencing the target gene (e.g., flanking smRNAsequences). Alternatively, the present disclosure also provides fordsRNA molecules having the second site located 5′ to the dsRNA sequencefor silencing the target gene, and the first site located 3′ to dsRNAsequence for silencing the target gene.

It will be appreciated that the complementary sequences can bepositioned on one strand (sense) and the other on the other strand(antisense). It will be further appreciated that in the presence of twocomplementary sequences to an smRNA or smRNAs, one of said sequences canmediate binding of the smRNA but not cleavage of the dsRNA sequence forsilencing the target gene. Thus, one of the complementary bindingsequences is essentially an smRNA mimic sequence (e.g. sufficient forbinding but not cleavage).

The smRNA mimic sequence is essentially complementary to the microRNA orsiRNA provided that one or more mismatches are allowed: thus, a mismatchbetween the complementary nucleotides at position 10 or position 11 ofthe microRNA and the corresponding nucleotide sequence in the micro-RNAresistant site. As used herein, the term “smRNA mimic,” “smRNA mutant,”and “miRNA Mut” are used interchangeably and refer to smRNAs that arenot cleaved in a cell. Not to be limited by any particular theory, mimicor mutant are thought mediate binding of the machinery, such as theArgonaute protein family, but are not processed, for example by adicer-like protein. Accordingly, smRNA mimics or mutants interact withthe RISC complex but can not be cleaved. Thus a non-cleavable targetmimic of a smRNA acts to sequester the corresponding target miRNA andarrest its activity. By incorporating a miRNA target mimic having anon-cleavable target site the accumulation of all MIR gene familymembers may be reduced. Methods for preparing smRNA mutants or mimicsare known. See Todesco et al., “A Collection of Target Mimics forComprehensive Analysis of MicroRNA Function in Arabidopsis thaliana,”PLOS Genetics 6(7):e1001031 (2010); Wang Z., “The guideline of thedesign and validation of MiRNA mimics,” Methods Mol Biol. 676:211-23(2011);

The complementary sequences may be identical or different and may berecognized by the same or different smRNAs, which may be miRNA or siRNA,or both, in any combination.

The complementary sequences(s) can be immediately adjacent to (i.e.,contiguous with) the dsRNA sequence for silencing the target gene.Alternatively, the complementary sequence (or at least one of same) maybe separated from the dsRNA sequence for silencing the target gene by anintervening spacer or functional sequence (e.g., Helicase binding site).

As used herein a “helicase binding site” refers to a binding site of anRNA helicase. RNA helicases are essential for most processes of RNAmetabolism such as ribosome biogenesis, pri-mRNA splicing andtranslation initiation. Sequence information is available from the RNAHelicase Database, available on the internet atwwwdotrnahelicasedotorg/. According to a specific embodiment, thehelicase may be a DEAD RNA helicase (DEAD RH), such as described in Chiet al. (2012). “The Function of RH22, a DEAD RNA Helicase, in theBiogenesis of the 50S Ribosomal Subunits of Arabidopsis Chloroplasts,”Plant Physiology, 158, 693-707. In certain embodiments, a helicasebinding site may be positioned so as to allow unwinding of the strandsof a dsRNA molecule to single stranded RNA (ssRNA) and allow recruitmentof an RNA-dependent RNA Polymerase such as RDR6. Unwinding andrecruitment of an RDRP provides for amplification of a dsRNA molecule inthe plant cell. Other proteins that are known to be cytosolic proteinsand have helicase or helicase-like activity include the Argonauteprotein family, which are a key components of the RISC complex (RNAInduced Silencing Complex). Alternatively a helicase binding site mayincludes sequences recognized by the plant homolog of RNA helicase RIG-I(Yoneyama et al. 2004 Nat. Immun. 5:730-737). Alternatively oradditionally, the present disclosure provides for helicase binding sitesequences as described in Garcia et al. 2012 Mol. Cell 48(1):109-20,which is hereby incorporated by reference in its entirety.

According to a specific embodiment, the helicase binding site ispositioned upstream or downstream of the dsRNA sequence for silencingexpression of the target gene (where the dsRNA sequence is referenced inthe antisense orientation).

According to a specific embodiment, the helicase binding site ispositioned upstream or downstream of the dsRNA sequence (where the dsRNAsequence is referenced in the antisense orientation) and the twocomplementary sites to the smRNA or smRNAs flank the helicase bindingsite.

According to a further specific embodiment, the helicase binding site islocated within the dsRNA sequence corresponding to the target site.

“Flanked by” as used herein, does not require that the smRNAcomplementary sequences are contiguous with the dsRNA sequence forsilencing the target gene. All that is necessary is that there is ansmRNA complementary sequences on at least one side or in case of twocomplementary sequences on each side of the dsRNA sequence for silencingthe target gene. Either or both smRNA complementary sequences may, invarious embodiments of the disclosure, be located contiguously with thedsRNA sequence for silencing the target gene. In certain embodimentseither or both smRNA complementary sequences may, in various embodimentsof the disclosure, be separated from the dsRNA sequence for silencingthe target gene by between 1 nt and 2 kB, e.g., between 1 nt and 1 kB,between 1 nt and 500 nt, between 1 nt and 250 nt, between 1 nt and 100nt, etc. In certain embodiments either or both smRNA complementarysequences are separated from a portion of the nucleic acid sequence thatcorresponds to the target gene by between 10 and 20 nt, between 10 and50 nt, or between 10 and 100 nt. Thus the spacer between either smRNAcomplementary sequences and the closest nucleotide that corresponds to aportion of a target gene may, in various embodiments of the disclosure,be between 1 nt and 2 kB, e.g., between 1 nt and 1 kB, between 1 nt and500 nt, between 1 nt and 250 nt, between 1 nt and 100 nt, between 10 and20 nt, between 10 and 50 nt, or between 10 and 100 nt in length.

Without being bound to theory, it is suggested that followingintroduction into the plant, the dsRNA molecule is unwound either by thebinding of a helicase to a “helicase binding site” when present or byendogenous RNA helicases that recognize and unwind the dsRNA molecule ina manner similar to antiviral response in a case where it is absent.

Without being bound to a particular theory, once a single strandedmolecule is formed is processed by miRNA-guided-cleavage. One product ofthe cleaved transcript may be stabilized possibly by Suppressor of GeneSilencing 3 (SGS3) and converted to dsRNA by RNA-Dependent RNAPolymerase 6 (RDR6). The resulting dsRNA may be processed throughDicer-Like 4 (DCL4) into 21-nt siRNA duplexes in register with themiRNA-cleavage site. One strand of each smRNA duplex may be selectivelysorted to one or more Argonaute (AGO) proteins according to the 5′nucleotide sequence while the other is used as a template for RNAdependent RNA polymerase, thereby constantly generating more phase siRNAmolecule.

Thus, the dsRNA molecule is designed for specifically targeting a targetgene of interest. It will be appreciated that the dsRNA can be used todown-regulate one or more target genes. In some embodiments, a singleisolated dsRNA molecule can target a number of different genes.

The present disclosure provides for and includes heterogeniccompositions of dsRNA molecules. In certain embodiments wherein a dsRNAmolecule targets a single target gene of interest, heterogeniccompositions comprising two or more dsRNA molecules that target two ormore target genes of interest may be prepared. A heterogenic compositioncomprises a plurality of dsRNA molecules for targeting a number oftarget genes may be prepared. In some embodiments, a plurality of dsRNAmolecules may be separately applied to the seeds (but not as a singlecomposition).

The present disclosure provides for an includes dsRNA moleculescomprising a sequence, wherein said nucleic acid sequence shares between100% and 90% sequence identity to a nucleic acid sequence selected fromthe group consisting of SEQ ID NO: 289-299. In other embodimentsaccording to the present disclosure, a dsRNA molecule comprises asequence that shares more than 90% sequence identity to a nucleic acidsequence selected from the group consisting of SEQ ID NO: 289-299. Inother embodiments according to the present disclosure, a dsRNA moleculecomprises a sequence that shares more than 91% sequence identity to anucleic acid sequence selected from the group consisting of SEQ ID NO:289-299. In other embodiments according to the present disclosure, adsRNA molecule comprises a sequence that shares more than 92% sequenceidentity to a nucleic acid sequence selected from the group consistingof SEQ ID NO: 289-299. In other embodiments according to the presentdisclosure, a dsRNA molecule comprises a sequence that shares more than93% sequence identity to a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 289-299. In other embodiments according to thepresent disclosure, a dsRNA molecule comprises a sequence that sharesmore than 94% sequence identity to a nucleic acid sequence selected fromthe group consisting of SEQ ID NO: 289-299. In other embodimentsaccording to the present disclosure, a dsRNA molecule comprises asequence that shares more than 95% sequence identity to a nucleic acidsequence selected from the group consisting of SEQ ID NO: 289-299. Inother embodiments according to the present disclosure, a dsRNA moleculecomprises a sequence that shares more than 96% sequence identity to anucleic acid sequence selected from the group consisting of SEQ ID NO:289-299. In other embodiments according to the present disclosure, adsRNA molecule comprises a sequence that shares more than 97% sequenceidentity to a nucleic acid sequence selected from the group consistingof SEQ ID NO: 289-299. In other embodiments according to the presentdisclosure, a dsRNA molecule comprises a sequence that shares more than98% sequence identity to a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 289-299. In other embodiments according to thepresent disclosure, a dsRNA molecule comprises a sequence that sharesmore than 99% sequence identity to a nucleic acid sequence selected fromthe group consisting of SEQ ID NO: 289-299. In other embodimentsaccording to the present disclosure, a dsRNA molecule comprises asequence that shares more than 100% sequence identity to a nucleic acidsequence selected from the group consisting of SEQ ID NO: 289-299.

While conceiving the present disclosure, the present inventors realizedthat the long dsRNA identified by Fukuhara et al. is able to surviveautonomously in rice cells, and as such can be used as a cassette (abuilding block) for introducing dsRNA sequenced for RNA silencing of atarget gene of interest (endogenous to the plant or exogenous thereto).Such a dsRNA molecule is expressed throughout the plant's life cycle,does not become integrated into the plant genome (plastid or nuclear),and does not get reverse-transcribed into DNA.

Thus, in some embodiments of the present disclosure provide forendovirus-derived sequences which have evolved to co-exist in plantcells in a dsRNA form, maintaining a near exact copy number in allcells.

As used herein the term “endovirus” refers to a dsRNA symbiotic viruswhich propagates in the plant cell and maintains a relatively stablecopy number throughout the life cycle of the plant.

As used herein the term “5′ UTR” refers to an untranslated regionderived from the endovirus sequence (13,716 nucleotides, available fromGSDB, DDBJ, EMBL, and NCBI nucleotide sequence data bases with accessionnumber D32136, according to Fukuhara 1995 supra), adjacent (in a 5′orientation) to its RDRP sequence.

As used herein the term “3′ UTR” refers to an untranslated regionderived from the endovirus sequence (13,716 nucleotides, available fromGSDB, DDBJ, EMBL, and NCBI nucleotide sequence data bases with accessionnumber D32136, according to Fukuhara 1995 supra), adjacent (in a 3′orientation) to its RDRP sequence.

As used herein the term “RNA dependent RNA Polymerase” refers to theRDRP-like sequence derived from the 13,716 nucleotides described inFukuhara et al. 1995 supra.

According to a specific embodiment said 5′ UTR is as set forth in SEQ IDNO: 14. It will be appreciated that the sequences are provided in theform of DNA but will be made RNA upon subjecting to T7 activity. In someembodiments, the 5′ UTR shares between 90% and 100% sequences identityto a nucleic acid sequence of SEQ ID NO:14.

According to a specific embodiment said 3′ UTR is as set forth in SEQ IDNO: 22. According to a specific embodiment said endovirus RNA DependentRNA Polymerase (RDRP) coding sequence is as set forth in SEQ ID NO: 23.

According to some embodiments of the disclosure, the nucleic acidsequence is at least about 80%, at least about 81%, at least about 82%,at least about 83%, at least about 84%, at least about 85%, at leastabout 86%, at least about 87%, at least about 88%, at least about 89%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, or more say100% identical to the nucleic acid sequence of SEQ ID NO: 14, 22 or 23(or a combination of same). It is understood that combinations of SEQ IDNOs: 14, 22, or 23 having differing percent homology are envisioned. Asa non-limiting example, a combination dsRNA molecule may comprise asequence having 85% homology to SEQ ID NO:14, 90% homology to SEQ IDNO:22 and 99% homology to SEQ ID NO:23. Any number of such combinationsare contemplated and provided for in the present disclosure.

One of ordinary skill in the art would understand that certainnucleotide positions of a polynucleotide sequence play critical roleswhile other nucleotide positions may vary without significant effect.This understanding is well illustrated for polypeptide encodingsequences. For example, the first and second nucleotide of an amino acidencoding codon largely determine the identity of an amino acid in apolypeptide chain. In contrast, the third “wobble” position can vary,sometimes without limitation. Accordingly, one of ordinary skill in theart can substitute approximately 30% of the nucleic acid sequencewithout affecting the amino acid sequence. A similar dependence fornon-coding sequences, including 3′ and 5′ UTRs exists, though thepositions of importance may not be predictable. However, one of ordinaryskill in the art may mutagenize (randomly or through site directed)positions in a nucleic acid sequence and confirm their activity usingthe functional assays disclosed in the present application (See,Examples below).

According to some embodiments, a nucleic acid sequence may encode aprotein that shares between 70% and 100% homology with an RNA DependentRNA Polymerase (RDRP) polypeptide sequence according to SEQ ID NO: 300.A nucleic acid sequence according to the present disclosure may encode aprotein having 70 to 75% homology to a polypeptide sequence according toSEQ ID NO: 300. In another embodiment, a nucleic acid sequence mayencode a protein having 75 to 80% homology to a polypeptide sequenceaccording to SEQ ID NO: 300. In another embodiment, a nucleic acidsequence may encode a protein having 80 to 85% homology to a polypeptidesequence according to SEQ ID NO: 300. In an embodiment, a nucleic acidsequence may encode a protein having 85 to 90% homology to a polypeptidesequence according to SEQ ID NO: 300. In another embodiment, a nucleicacid sequence may encode a protein having 90 to 95% homology to apolypeptide sequence according to SEQ ID NO: 300. In an embodiment, anucleic acid sequence may encode a protein having 95 to 100% homology toa polypeptide sequence according to SEQ ID NO: 300. In anotherembodiment, a nucleic acid sequence may encode a protein having 97 to100% homology to a polypeptide sequence according to SEQ ID NO: 300. Inan embodiment, a nucleic acid sequence may encode a protein having 98 to100% homology to a polypeptide sequence according to SEQ ID NO: 300. Inanother embodiment, a nucleic acid sequence may encode a protein having99 to 100% homology to a polypeptide sequence according to SEQ ID NO:300.

Homologous sequences to the above can also be used according to thepresent teachings, as long as their main characteristics are maintained,i.e., amplification by RDRP and maintenance of stable copy number in thecell throughout the plant life cycle.

The present disclosure further provides for, and includes, an isolateddsRNA molecule comprising a nucleic acid sequence in a sequential orderfrom 5′ to 3′, an endovirus 5′ UTR, an endovirus RNA Dependent RNAPolymerase (RDRP) coding sequence, an endovirus 3′ UTR and a cloningsite flanked by said RDRP and said 3′ UTR.

The present disclosure further provides for, and includes, an isolateddsRNA molecule comprising a nucleic acid sequence in a sequential orderfrom 5′ to 3′, an endovirus 5′ UTR, an endovirus RNA Dependent RNAPolymerase (RDRP) coding sequence, an endovirus 3′ UTR and a nucleicacid sequence for regulating a target gene flanked by said RDRP and said3′ UTR.

In some embodiments, a heterologous dsRNA sequence corresponding to atarget gene is constructed such that it is flanked by the RDRP sequenceand the 3′ UTR. Alternatively, in other embodiments, a heterologousdsRNA sequence corresponding to the target gene is constructed such thatit is flanked by the RDRP sequence and the 5′ UTR. When introduced intothe plant (e.g., directly to the seed), and once germination hasinitiated, gene expression occurs including expression of endogenousplant helicases, RDRPs and other components of the silencing machinery.Not to be limited by theory, at any given time, a portion of the dsRNAmolecules is recognized and processed by the plant's dicer like (DCL)proteins into siRNA of different lengths. In certain embodiments, thisrecognition and processing includes processing of the gene targeted forsilencing, which is flanked between the 5′ and the 3′ UTR. In certainembodiments, the gene targeted for silencing and the RDRP sequence isflanked between the 5′ and the 3′ UTR. Not all of the heterologous dsRNAsequence is processed and a portion remains in double-stranded, fulllength form. Not to be limited by theory, it is thought that planthelicases recognize unique features in the 5′ UTR and the 3′ UTR of thedsRNA and unwind the dsRNA into two ssRNA molecules. Again, not limitedby theory, the same or other feature in the 5′ and 3′ UTR are thought toalso recruit and activate an RDRP. In some embodiments, the RDRP may bean RDRP that is encoded by the dsRNA. In other embodiments, the RDRP maybe an endogenous RDRP, naturally occurring in the plant or introduced asa transgene.

Though not limited by theory, it is thought that the RDRP uses each ofthe ssRNA molecules thought to be produced by the activity of a helicaseas templates to produce a dsRNA molecule identical to the original dsRNAmolecule.

Accordingly, and not to be limited by theory, as long as a ratio betweenprocessed and un-processed dsRNA molecules is maintained, the cycle cango on and repeat throughout the plant's life cycle. Also not to belimited by theory, it is thought that some features in the 5′ and 3′ UTRcan assist to maintain a stable copy number of the dsRNA in cells, in asimilar manner to the endovirus which is maintained at a stable copynumber.

As used herein the term “sequential” refers to multiple nucleic acidsegments (e.g., 5′ UTR, RDRP and 3′ UTR) arranged in sequence. In thiscase, from 5′ to 3. Each of the specified nucleic acid segments can bedirectly attached to each other and contiguous, however interveningnucleic acids can be implanted there between such that the segments arenot directly attached to each other and are discontinuous.

According to an embodiment of the disclosure the target gene isendogenous to the plant. Downregulating such a gene is typicallyimportant for conferring the plant with an improved, agricultural,horticultural, nutritional trait (“improvement” or an “increase” isfurther defined hereinbelow).

As used herein, the terms “suppress,” “repress,” and “downregulate” whenreferring to the expression or activity of a nucleic acid molecule in aplant cell are used equivalently herein and mean that the level ofexpression or activity of the nucleic acid molecule in a plant, a plantpart, or plant cell after applying a method of the present disclosure islower than its expression or activity in the plant, part of the plant,or plant cell before applying the method, or compared to a control plantlacking a dsRNA molecule of the disclosure.

The terms “suppressed,” “repressed” and “downregulated” as used hereinare synonymous and mean herein lower, preferably significantly lower,expression or activity of the nucleic acid molecule to be expressed.

As used herein, a “suppression,” “repression,” or “downregulation” ofthe level or activity of an agent such as a protein, mRNA, or RNA meansthat the level or activity is reduced relative to a substantiallyidentical plant, part of a plant, or plant cell grown undersubstantially identical conditions, lacking a dsRNA molecule of thedisclosure, for example, lacking an RNA sequence for regulating a targetgene of interest. As used herein, “suppression,” “repression,” or“downregulation” of the level or activity of an agent, such as, forexample, a preRNA, mRNA, rRNA, tRNA, snoRNA, snRNA expressed by thetarget gene, and/or of the protein product encoded by it, means that theamount is reduced by 10% or more, for example, 20% or more, preferably30% or more, more preferably 50% or more, even more preferably 70% ormore, most preferably 80% or more, for example, 90%, relative to a cellor organism lacking a dsRNA molecule of the disclosure.

As used herein “a suppressive amount” refers to an amount of dsRNAmolecule which is sufficient to down regulate the target gene by atleast 20%. In an aspect, a suppressive amount according to the presentdisclosure is an amount sufficient to downregulate a target gene by 30%or more. In an aspect, a suppressive amount according to the presentdisclosure is an amount sufficient to downregulate a target gene by 40%or more. In an aspect, a suppressive amount according to the presentdisclosure is an amount sufficient to downregulate a target gene by atleast 50%. In other aspects, a suppressive amount according to thepresent disclosure is an amount sufficient to downregulate a target geneby 60% or more. In an aspect, a suppressive amount according to thepresent disclosure is an amount sufficient to downregulate a target geneby at least 70%. In an aspect, a suppressive amount according to thepresent disclosure is an amount sufficient to downregulate a target geneby 80% or more. In an aspect, a suppressive amount according to thepresent disclosure is an amount sufficient to downregulate a target geneby greater than 90%. In certain aspects, a suppressive amount accordingto the present disclosure is an amount sufficient to downregulate atarget gene by 100% (e.g., wherein the remaining amount of the targetgene is not detectable). The suppressive amount can be a result of theformation of amplification in the plant or the phytopathogen.

As used herein “endogenous” refers to a gene which expression (mRNA orprotein) takes place in the plant. Typically, the endogenous gene isnaturally expressed in the plant or originates from the plant. Thus, theplant may be a wild-type plant. However, the plant may also be agenetically modified plant (transgenic).

Downregulation of the target gene may be important for conferringimproved one of-, or at least one of (e.g., two of- or more), biomass,vigor, yield, abiotic stress tolerance, biotic stress tolerance orimproved nitrogen use efficiency.

Examples of target genes include, but are not limited to, an enzyme, astructural protein, a plant regulatory protein, an miRNA target gene, ora non-coding RNA such as a miRNA of the plant. WO2011067745, WO2009125401 and WO 2012056401 provide examples of miRNA sequences ortargets of miRNAs (e.g., miRNA167, miRNA169, miRNA 156, miR164 andtargets thereof ARF, NFY, SPL17 and NAC, respectively) which expressioncan be silenced to improve a plant trait.

The target gene may comprise a nucleic acid sequence which istranscribed to an mRNA which codes for a polypeptide.

Alternatively, the target gene can be a non-coding gene such as an miRNAor a siRNA.

For example, in order to silence the expression of an mRNA of interest,synthesis of the dsRNA molecule suitable for use with some embodimentsof the disclosure can be selected as follows. First, the mRNA sequencemay be scanned including the 3′ UTR and the 5′ UTR.

Second, the mRNA sequence may be compared to an appropriate genomicdatabase using any sequence alignment software, such as the BLASTsoftware available from the NCBI server (available on the internet atwwwdotncbidotnlmdotnihdotgov/BLAST/). Putative regions in the mRNAsequence which exhibit significant homology to other coding sequencesmay be filtered out.

Qualifying target sequences may be selected as for the preparation ofdsRNA templates for dsRNA molecule synthesis. Preferred sequences arethose that have as little homology to other genes in the genome toreduce an “off-target” effect.

It will be appreciated that the RNA regulating or silencing agent ofsome embodiments of the disclosure need not be limited to thosemolecules containing only RNA, but further encompasseschemically-modified nucleotides and non-nucleotides.

The dsRNA molecules of the present disclosure may be synthesized usingany method known in the art, including either enzymatic syntheses orsolid-phase syntheses. These are especially useful in the case of shortpolynucleotide sequences with or without modifications as explainedabove. Equipment and reagents for executing solid-phase synthesis arecommercially available from, for example, Applied Biosystems. Any othermeans for such synthesis may also be employed; the actual synthesis ofthe oligonucleotides is well within the capabilities of one skilled inthe art and can be accomplished via established methodologies asdetailed in, for example: Sambrook, J. and Russell, D. W. (2001),“Molecular Cloning: A Laboratory Manual”; Ausubel, R. M. et al., eds.(1994, 1989), “Current Protocols in Molecular Biology,” Volumes I-III,John Wiley & Sons, Baltimore, Md.; Perbal, B. (1988), “A Practical Guideto Molecular Cloning”, John Wiley & Sons, New York; and Gait, M. J., ed.(1984), “Oligonucleotide Synthesis”; utilizing solid-phase chemistry,e.g., cyanoethyl phosphoramidite followed by deprotection, desalting,and purification by, for example, an automated trityl-on method or HPLC.

The nucleic acids of the present disclosure may comprise heterocylicnucleosides consisting of purines and the pyrimidines bases, bonded in a3′ to 5′ 5phosphodiester linkage. Preferably used nucleic acids arethose modified in either backbone, internucleoside linkages or bases, asis broadly described hereinunder.

Specific examples of preferred nucleic acids useful according to thisaspect of the present disclosure include nucleic acids containingmodified backbones or non-natural internucleoside linkages. Nucleicacids having modified backbones include those that retain a phosphorusatom in the backbone, as disclosed in U.S. Pat. Nos. 4,469,863;4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306;5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

Preferred modified polynucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms can also be used.

Alternatively, modified polynucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,0, S and CH2 component parts, as disclosed in U.S. Pat. Nos. 5,034,506;5,166,315; 5,185,444; 5,216,141; 5,235,033; 5,264,562; 5,264,564;5,405,938; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086;5,602,240; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070;5,663,312; 5,214,134; 5,466,677; 5,610,289; 5,633,360; 5,677,437; and5,677,439.

Other nucleic acids which can be used according to the presentdisclosure, are those modified in both sugar and the internucleosidelinkage, i.e., the backbone, of the nucleotide units are replaced withnovel groups. The base units are maintained for complementation with theappropriate polynucleotide target. An example for such an polynucleotidemimetic, includes peptide nucleic acid (PNA). A PNA polynucleotiderefers to a polynucleotide where the sugar-backbone is replaced with anamide containing backbone, in particular an aminoethylglycine backbone.The bases are retained and are bound directly or indirectly to azanitrogen atoms of the amide portion of the backbone. United Statespatents that teach the preparation of PNA compounds include, but are notlimited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each ofwhich is herein incorporated by reference. Other backbone modifications,which can be used in the present disclosure are disclosed in U.S. Pat.No. 6,303,374.

Polynucleotide agents of the present disclosure may also include basemodifications or substitutions. As used herein, “unmodified” or“natural” bases include the purine bases adenine (A) and guanine (G),and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).Modified bases include but are not limited to other synthetic andnatural bases such as 5-methyl cytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.Further bases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 613, and those disclosed by Sanghvi, Y. S.,Chapter 15, Antisense Research and Applications, pages 289-2, Crooke, S.T. and Lebleu, B., ed., CRC Press, 1993. Such bases are particularlyuseful for increasing the binding affinity of the oligomeric compoundsof the disclosure. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi Y S et al. (1993) AntisenseResearch and Applications, CRC Press, Boca Raton 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

Following synthesis, the polynucleotide agents of the present disclosuremay optionally be purified. For example, polynucleotides can be purifiedfrom a mixture by extraction with a solvent or resin, precipitation,electrophoresis, chromatography, or a combination thereof.Alternatively, polynucleotides may be used with no, or a minimum of,purification to avoid losses due to sample processing. Thepolynucleotides may be dried for storage or dissolved in an aqueoussolution. The solution may contain buffers or salts to promoteannealing, and/or stabilization of the duplex strands.

It will be appreciated that a polynucleotide agent of the presentdisclosure may be provided per se, or as a nucleic acid constructcomprising a nucleic acid sequence encoding the polynucleotide agent.Typically, the nucleic acid construct comprises a promoter sequencewhich is functional in the host cell, as detailed herein below.

The polynucleotide sequences of the present disclosure, under thecontrol of an operably linked promoter sequence, may further be flankedby additional sequences that advantageously affect its transcriptionand/or the stability of a resulting transcript. Such sequences aregenerally located upstream of the promoter and/or downstream of the 3′end of the expression construct.

The term “operably linked”, as used in reference to a regulatorysequence and a structural nucleotide sequence, means that the regulatorysequence causes regulated expression of the linked structural nucleotidesequence. “Regulatory sequences” or “control elements” refer tonucleotide sequences located upstream, within, or downstream of astructural nucleotide sequence, and which influence the timing and levelor amount of transcription, RNA processing or stability, or translationof the associated structural nucleotide sequence. Regulatory sequencesmay include promoters, translation leader sequences, introns, enhancers,stem-loop structures, repressor binding sequences, terminationsequences, pausing sequences, polyadenylation recognition sequences, andthe like.

The present disclosure provides for and includes DNA templates for thepreparation of dsRNA molecules. As used herein, “dsRNA template” refersto a DNA sequence having the same sequence as the corresponding dsRNAmolecule. In certain embodiments, a dsRNA template may further includeadditional sequences, such as promoters, sufficient for the expressionof one or more RNA molecules via transcription. In some embodiments thepromoters are bacteriophage promoters, for example but not limited to,SP6, T3 and T7. In some embodiments a promoter may be a bacterial or aeukaryotic promoters.

In certain embodiments, a dsRNA template according to the presentteachings may be used as a cassette for the cloning of a nucleic acidsequence corresponding to a target gene of interest (exogenous to theplant or endogenous thereto) for silencing expression of same whenexpressed as a dsRNA molecule.

Thus according to an embodiment of the disclosure, a dsRNA template maycomprise a cloning site (multiple cloning site for instance) to which anucleic sequence for silencing a target gene of interest is ligatedwhile being flanked by the RDRP encoding and the 3′UTR encoding nucleicacid sequences. In other aspects according to present disclosure, adsRNA molecule may be prepared by chemical synthesis using methods knownin the art. In other embodiments, a dsRNA template may comprise aplasmid vector having the dsRNA molecule coding sequences. In certainembodiments, the dsRNA template may be a linear polynucleotide having aRNA polymerase promoter at one end. It will be appreciated that such atemplate produces a single strand RNA.

In certain embodiments, a dsRNA template may comprise a mixture of twolinear polynucleotides having the same dsRNA coding sequence butpromoters at opposite ends. It will be appreciated that transcriptionresults in the production of two complementary RNA strands from theseparate template that may be annealed and recovered, or recovered andannealed. By providing separate transcription templates, and recoveringannealed double stranded dsRNA molecules according to the presentdisclosure, asymmetric dsRNA molecules may be produced. As providedbelow, dsRNA construct #4 provides for a dsRNA region corresponding to aregion of a target gene of interest and having non-double strandedMir390 Mut BS and Mir390BS sequences. As used herein, the term“overhang” refers to non-double stranded regions of a dsRNA molecule(i.e., single stranded RNA). Accordingly dsRNA construct #4 has twooverhang regions comprising Mir390 Mut BS and Mir390 BS sequencesrespectively. Similarly, dsRNA construct #6 provides for an asymmetricdsRNA having a non-double stranded helicase binding sequence oroverhang.

In other aspects, a dsRNA template may have two promoters flanking thedsRNA coding sequences. It will be appreciated that, like the separatetemplates, two complementary strands are produced that may be annealedand recovered, or recovered and annealed. The promoters of the dsRNAtemplates of some embodiments may be the same or different.

As mentioned, in certain embodiments, the dsRNA molecule may be directlycontacted with the seed.

The seed may be of any plant, such as of the Viridiplantae super familyincluding monocotyledon and dicotyledon plants. Other plants are listedbelow. According to an embodiment of the disclosure, the cells of theplant comprise RNA dependent RNA polymerase activity and the target RNAmolecule of the dsRNA to ensure amplification of the dsRNA.

The term “plant” as used herein encompasses whole plants, ancestors andprogeny of the plants and plant parts, including seeds, shoots, stems,roots (including tubers), and isolated plant cells, tissues and organs.The plant may be in any form including suspension cultures, embryos,meristematic regions, callus tissue, leaves, gametophytes, sporophytes,pollen, and microspores. It will be appreciated, that the plant or seedthereof may be transgenic plants.

As used herein the phrase “plant cell” refers to plant cells which arederived and isolated from disintegrated plant cell tissue or plant cellcultures.

As used herein the phrase “plant cell culture” refers to any type ofnative (naturally occurring) plant cells, plant cell lines andgenetically modified plant cells, which are not assembled to form acomplete plant, such that at least one biological structure of a plantis not present. Optionally, the plant cell culture of this embodiment ofthe present disclosure may comprise a particular type of a plant cell ora plurality of different types of plant cells. It should be noted thatoptionally plant cultures featuring a particular type of plant cell maybe originally derived from a plurality of different types of such plantcells. In certain embodiments according to the present disclosure, theplant cell is a non-sexually producing plant cell. In other aspects, aplant cell of the present disclosure is a non-photosynthetic plant cell.

Any commercially or scientifically valuable plant is envisaged inaccordance with some embodiments of the disclosure. Plants that areparticularly useful in the methods of the disclosure include all plantswhich belong to the super family Viridiplantae, in particularmonocotyledonous and dicotyledonous plants including a fodder or foragelegume, ornamental plant, food crop, tree, or shrub selected from thelist comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp.,Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp.,Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaeaplurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkeaafricana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camelliasinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens,Chacoomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermummopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumisspp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeriajaponica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergiamonetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa,Dibeteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum,Echinochloa pyramidalis, Ehraffia spp., Eleusine coracana, Eragrestisspp., Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulaliavi/losa, Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingiaspp, Freycinetia banksli, Geranium thunbergii, GinAgo biloba, Glycinejavanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtiacoleosperma, Hedysarum spp., Hemaffhia alassima, Heteropogon contoffus,Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffheliadissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia,Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex,Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihotesculenta, Medicago saliva, Metasequoia glyptostroboides, Musasapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryzaspp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petuniaspp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photiniaspp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara,Pogonarthria fleckii, Pogonaffhria squarrosa, Populus spp., Prosopiscineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis,Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhusnatalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosaspp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitysvefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghumbicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides,Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themedatriandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vacciniumspp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschiaaethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brusselssprouts, cabbage, canola, carrot, cauliflower, celery, collard greens,flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean,straw, sugar beet, sugar cane, sunflower, tomato, squash tea, maize,wheat, barley, rye, oat, peanut, pea, lentil and alfalfa, cotton,rapeseed, canola, pepper, sunflower, tobacco, eggplant, eucalyptus, atree, an ornamental plant, a perennial grass and a forage crop.Alternatively algae and other non-Viridiplantae can be used for themethods of the present disclosure.

According to some embodiments of the disclosure, the plant used by themethod of the disclosure is a crop plant including, but not limited to,cotton, Brassica vegetables, oilseed rape, sesame, olive tree, palm oil,banana, wheat, corn or maize, barley, alfalfa, peanuts, sunflowers,rice, oats, sugarcane, soybean, turf grasses, barley, rye, sorghum,sugar cane, chicory, lettuce, tomato, zucchini, bell pepper, eggplant,cucumber, melon, watermelon, beans, hibiscus, okra, apple, rose,strawberry, chili, garlic, pea, lentil, canola, mums, Arabidopsis,broccoli, cabbage, beet, quinoa, spinach, squash, onion, leek, tobacco,potato, sugar beet, papaya, pineapple, mango, Arabidopsis thaliana, andalso plants used in horticulture, floriculture or forestry, such as, butnot limited to, poplar, fir, eucalyptus, pine, an ornamental plant, aperennial grass and a forage crop, coniferous plants, moss, algae, aswell as other plants available on the internet at, for example,wwwdotnationmasterdotcom/encyclopedia/Plantae.

According to a specific embodiment, the plant is selected from the groupconsisting of corn, rice, wheat, tomato, cotton and sorghum. In certainembodiments, the plant is a corn plant. In certain embodiments, theplant is a rice plant. In certain embodiments, the plant is a wheatplant. In certain embodiments, the plant is a cotton plant. In certainembodiments, the plant is a sorghum plant.

Introduction of the compositions of the present disclosure can beperformed to any organs/cells of the plant (as opposed to seeds) usingconventional delivery methods such as particle bombardment, grafting,soaking and the like.

According to a specific embodiment, the composition is introduceddirectly to the seed. According to a specific embodiment, the seed is anuncoated or fresh seed that hasn't been subjected to chemical/physicaltreatments. In certain embodiments, the seed is a corn seed. In certainembodiments, the seed is a rice seed. In certain embodiments, the seedis a wheat seed. In certain embodiments, the seed is a cotton seed. Incertain embodiments, the seed is a sorghum seed.

The seed may be subjected to priming or washing prior to contacting withthe dsRNA.

In embodiments according to the present disclosure, washing of the seedsis effected for 30 min to 4 hours. In other embodiments, the seeds maybe washed up to 5 hours. In an embodiment a seed may be washed up 6, 7or 8 hours. In another embodiment, seeds are washed for less than 4 orless than 3 hours. In some embodiments, a seed may be washed less thantwo hours or less than one hour.

The present disclosure provides for and includes washing of a seedbetween 1 minute and 1 hours. Also included are brief washes comprisingless than 1 minute wherein the seed is completely wet and then removedfrom the wash. In some embodiments, a seed may be washed from 1 minuteto 10 minutes. In another embodiment, a seed may be washed from 1 minuteto 10 minutes. In an embodiment, a seed may be washed from 10 to 30minutes. In yet another embodiment, a seed may be washed from 1 to 30minutes. In certain embodiments, a seed may be washed from 5 to 10minutes or 5 to 15 minutes. In some embodiments, a seed may be washedfrom 15 to 30 minutes or 10 to 25 minutes.

In some embodiments according to the present disclosure, the washsolution may include a weak detergent such as Tween-20, or itsequivalents. In some embodiments, the detergent may be less than 1% byvolume. In other embodiments, the detergent may be less than 0.5% byvolume. In some embodiments, the detergent may be less than 0.25% or0.2% by volume. In other embodiments, the detergent may be less than0.1% or 0.05% by volume. In embodiments according to the presentdisclosure, the wash solution may contain a detergent at between 0.01 to0.2% or 0.2 to 1%. In other embodiments, the wash solution may contain adetergent at between 0.05 to 0.5% or 0.5 to 1.5%.

As used herein the term “priming” refers to controlling the hydrationlevel within seeds so that the metabolic activity necessary forgermination can occur but radicle emergence is prevented. Differentphysiological activities within the seed occur at different moisturelevels (Leopold and Vertucci, 1989; Taylor, 1997). The lastphysiological activity in the germination process is radicle emergence.The initiation of radicle emergence requires a high seed water content.By limiting seed water content, all the metabolic steps necessary forgermination can occur without the irreversible act of radicle emergence.Prior to radicle emergence, the seed is considered desiccation tolerant,thus the primed seed moisture content can be decreased by drying. Afterdrying, primed seeds can be stored until time of sowing.

Several different priming methods are used commercially. Among them,liquid or osmotic priming and solid matrix priming appear to have thegreatest following (Khan et al., 1991).

According to an embodiment of the disclosure, priming is effected in thepresence of salt, a chelating agent, polyethylene glycol or acombination of same (e.g., chelating agent and salt).

Alternatively priming is effected in the presence of water such asdeionized water or double deionized water (ddW). According to a specificembodiment, the priming is effected in the presence of 100% ddW.

Several types of seed priming are commonly used:

Osmopriming (osmoconditioning) is a standard priming technique. Seedsare incubated in well aerated solutions with a low water potential, andafterwards washes and dried. The low water potential of the solutionscan be achieved by adding osmotica like mannitol, polyethyleneglycol(PEG) or salts like KCl. In embodiments according to the presentdisclosure, the seeds are osmoprimed. In certain embodiments, theosmoprimed seed is a corn seed. In certain embodiments, the osmoprimedseed is a rice seed. In certain embodiments, the osmoprimed seed is awheat seed. In certain embodiments, the osmoprimed seed is a cottonseed. In certain embodiments, the osmoprimed seed is a sorghum seed.

Hydropriming (drum priming) is achieved by continuous or successiveaddition of a limited amount of water to the seeds. A drum is used forthis purpose and the water can also be applied by humid air. ‘On-farmsteeping’ is a cheap and useful technique that is practiced byincubating seeds (cereals, legumes) for a limited time in warm water. Inembodiments according to the present disclosure, the seeds arehydroprimed. In certain embodiments, the hydroprimed seed is a cornseed. In certain embodiments, the hydroprimed seed is a rice seed. Incertain embodiments, the hydroprimed seed is a wheat seed. In certainembodiments, the hydroprimed seed is a cotton seed. In certainembodiments, the hydroprimed seed is a sorghum seed.

Matrixpriming (matriconditioning) is the incubation of seeds in a solid,insoluble matrix (vermiculite, diatomaceous earth, cross-linked highlywater-absorbent polymers) with a limited amount of water. This methodconfers a slow imbibition. In embodiments according to the presentdisclosure, the seeds are matriconditioned. In certain embodiments, thematriconditioned seed is a corn seed. In certain embodiments, thematriconditioned seed is a rice seed. In certain embodiments, thematriconditioned seed is a wheat seed. In certain embodiments, thematriconditioned seed is a cotton seed. In certain embodiments, thematriconditioned seed is a sorghum seed.

Pregerminated seeds may be used in certain embodiments however not allspecies can be primed using this method. In contrast to normal priming,seeds are allowed to perform radicle protrusion. This is followed bysorting for specific stages, a treatment that reinduces desiccationtolerance, and drying. The use of pregerminated seeds causes rapid anduniform seedling development.

Thus, according to one embodiment, the seeds are primed seeds.

Of note, it may be possible that the seeds are treated with water(double-distilled water, ddW), prior to contacting with the dsRNAwithout effecting any priming on the seeds. For instance, treatment fora short while with water (e.g., 30 seconds to 1 hours, 30 seconds to 0.5hour, 30 seconds to 10 min, 30 seconds to 5 min or 45 seconds to 5 min).

Thus, according to one embodiment, the seeds are non-primed seeds.

A non-limiting example of a method of introducing the dsRNA into theseed is provided in Example 1, which is considered as an integral partof the specification.

The temperature at the washing/priming and drying steps may be the sameor differ.

According to one embodiment, the temperature for washing/priming isbetween 4 and 28° C. In some embodiments, the washing/primingtemperature is less than 28° C. In some embodiments, the washing/primingtemperature is less than 25° C. In some embodiments, the washing/primingtemperature is less than 20° C. In some embodiments, the washing/primingtemperature is less than 15° C. In some embodiments, the washing/primingtemperature is less than 10° C. In some embodiments, the washing/primingtemperature is between 4 and 10° C. In an embodiment the washing/primingtemperature is between 10 and 15° C. In an another embodiment thewashing/priming temperature is between 15 and 20° C. or 15 and 25° C. Inan another embodiment the washing/priming temperature is between 10 and20° C. or 10 and 25° C.

According to one embodiment, the priming/washing solution or the dsRNAcontaining solution is devoid of a solid carrier.

According to one embodiment, the priming/washing solution or the dsRNAcontaining solution is devoid of a transferring agent such as asurfactant or a salt.

According to a further embodiment of the disclosure, the seeds subjectto contacting with the dsRNA molecule are washed in order to removeagents, to which the seeds have been subjected, such as a pesticide, afungicide, an insecticide, a fertilizer, a coating agent and a coloringagent.

Thus, according to one embodiment, the seeds (prior to treatment withdsRNA) are substantially free (i.e., do not comprise effective amounts)of pesticide, a fungicide, an insecticide, a fertilizer, a coating agentand a coloring agent.

The seeds are then subjected to drying.

According to one embodiment, the drying temperature is between 20 to 37°C. In another embodiment, the drying temperature is between 20 to 30° C.In another embodiment, the drying temperature is between 22 and 37° C.In another embodiment, the drying temperature is between 15 to 22° C. or20 to 25° C. In embodiments according to the present disclosure, thedrying time at a temperature of the present disclosure may be for 10 to20 hours. In other embodiments the drying time may be from 10 to 16hours. In other embodiments, the drying time may be 2 to 5 hours.

Various considerations are to be taken when calculating theconcentration of the dsRNA in the contacting solution. Considerationsinclude, but are not limited to, at least one of the group consisting ofseed size, seed weight, seed volume, seed surface area, seed density andseed permeability.

For example, related to seed size, weight, volume and surface area, itis estimated that corn seeds will require longer treatment thanArabidopsis and tomato seeds. Regarding permeability and density, it isestimated that wheat seeds will require longer treatments at higherconcentrations than tomato seeds.

Examples of concentrations of dsRNA in the treating solution include,but are not limited to 0.1 to 100 micrograms (1×10⁻⁶ grams) permicroliter (1×10⁻⁶ liter) (μg/μl). In an embodiment the dsRNAconcentration in the treating solution may be 0.04 to 0.15 μg/μl. In ananother embodiment the dsRNA concentration in the treating solution maybe 0.1 to 50 μg/μl. In certain embodiments, the dsRNA concentration inthe treating solution may be 0.1 to 10 μg/μl. In yet other embodimentsthe dsRNA concentration in the treating solution may be 0.1 to 5 μg/μl.In some embodiments the dsRNA concentration in the treating solution maybe 0.1 to 1 μg/μl. In an embodiment the dsRNA concentration in thetreating solution may be 0.1 to 0.5 μg/μl. Also included and providedfor in the present disclosure are embodiments having a dsRNAconcentration in the treating solution of between 0.15 and 0.5 μg/μl. Inan embodiment the dsRNA concentration in the treating solution may be0.1 to 0.3 μg/μl. In an embodiment the dsRNA concentration in thetreating solution may be 0.01 to 0.1 μg/μl. In an embodiment the dsRNAconcentration in the treating solution may be 0.01 to 0.05 μg/μl. In anembodiment the dsRNA concentration in the treating solution may be 0.02to 0.04 μg/μl. In an embodiment the dsRNA concentration in the treatingsolution may be 0.001 to 0.02 1.1 g/μl. According to a specificembodiment, the concentration of the dsRNA in the treating solution is0.04 to 0.15 μg/μl.

According to a specific embodiment, the contacting with the dsRNA iseffected in the presence of a chelating agent such as EDTA or anotherchelating agent such as DTPA (0.01 to 0.1 mM).

The contacting solution may comprise a transferring agent such as asurfactant or a salt.

Examples of such transferring agents include but are not limited saltssuch as sodium or lithium salts of fatty acids (such as tallow ortallowamines or phospholipids lipofectamine or lipofectin (1 to 20 nM,or 0.1 to 1 nM)) and organosilicone surfactants. Other usefulsurfactants include organosilicone surfactants including nonionicorganosilicone surfactants, e.g., trisiloxane ethoxylate surfactants ora silicone polyether copolymer such as a copolymer of polyalkylene oxidemodified heptamethyl trisiloxane and allyloxypolypropylene glycolmethylether (commercially available as Silwet™ L-77 surfactant havingCAS Number 27306-78-1 and EPA Number: CAL.REG.NO. 5905-50073-AA,currently available from Momentive Performance Materials, Albany, N.Y.).

Useful physical agents can include (a) abrasives such as carborundum,corundum, sand, calcite, pumice, garnet, and the like, (b) nanoparticlessuch as carbon nanotubes or (c) a physical force. Carbon nanotubes aredisclosed by Kam et al. (2004) J. Am. Chem. Soc., 126 (22):68506851, Liuet al. (2009) Nano Lett., 9(3):1007-1010, and Khodakovskaya et al.(2009) ACS Nano, 3(10):3221-3227. Physical force agents can includeheating, chilling, the application of positive pressure, or ultrasoundtreatment. Agents for laboratory conditioning of a plant to permeationby polynucleotides include, e.g., application of a chemical agent,enzymatic treatment, heating or chilling, treatment with positive ornegative pressure, or ultrasound treatment. Agents for conditioningplants in a field include chemical agents such as surfactants and salts.

Contacting of the seeds with the dsRNA molecule can be effected usingany method known in the art as long as a suppressive amount of the dsRNAmolecule enters the seeds. These examples include, but are not limitedto, soaking, spraying or coating with powder, emulsion, suspension, orsolution; similarly, the polynucleotide molecules are applied to theplant by any convenient method, e.g., spraying or wiping a solution,emulsion, or suspension.

According to a specific embodiment contacting may be effected by soaking(i.e., inoculation) so that shaking the seeds with the treating solutionmay improve penetration and soaking and therefore reduce treatment time.Shaking is typically performed at 50 to 150 RPM and depends on thevolume of the treating solution. Shaking may be effected for 4 to 24hours (1 to 4 hours, 10 minutes to 1 hour or 30 seconds to 10 minutes).The incubation takes place in the dark at 4 to 28° C. or 15 to 22° C.(e.g., 8 to 15° C., 4 to 8° C., 22 to 28° C.).

According to a specific embodiment, contacting occurs prior to breakingof seed dormancy and embryo emergence.

Following contacting, preferably prior to breaking of seed dormancy andembryo emergence, the seeds may be subjected to treatments (e.g.,coating) with the above agents (e.g., pesticide, fungicide etc.).

Contacting is effected such that the dsRNA molecule enters the embryo,endosperm, the coat, or a combination of the three.

After contacting with the treatment solution, the seeds may be subjectedto drying for up to 30 hours at 25 to 37° C.

According to a specific embodiment, the seed (e.g., isolated seed)comprises the exogenous dsRNA and wherein at least 10 or 20 molecules ofthe dsRNA are in the endosperm of the isolated seed.

As used herein the term “isolated” refers to separation from the naturalphysiological environment. In the case of seed, the isolated seed isseparated from other parts of the plant. In the case of a nucleic acidmolecule (e.g., dsRNA) separated from the cytoplasm.

According to a specific embodiment, the dsRNA molecule is not expressedfrom the plant genome, thereby not being an integral part of the genome.

Methods of qualifying successful introduction of the dsRNA moleculeinclude but are not limited to, RT-PCR (e.g., quantifying the level ofthe target gene or the dsRNA), phenotypic analysis such as biomass,vigor, yield and stress tolerance, root architecture, leaf dimensions,grain size and weight, oil content, cellulose, as well as cell biologytechniques.

Seeds may be stored for 1 day to several months prior to planting (e.g.,at 4 to 10° C.).

The resultant seed can be germinated in the dark so as to produce aplant.

Thus there is provided a plant or plant part comprising an exogenousdsRNA molecule and devoid of a heterologous promoter for drivingexpression of the dsRNA molecule in the plant.

As used herein “devoid of a heterologous promoter for driving expressionof the dsRNA” means that the plant or plant cell doesn't include acis-acting regulatory sequence (e.g., heterologous) transcribing thedsRNA in the plant.

As used herein, the term “heterologous” means not naturally occurringtogether. In some embodiments, the term “heterologous” refers toexogenous, not-naturally occurring within the native plant cell (such asby position of integration, or being non-naturally found within theplant cell). Thus the isolated seed in the absence of a heterologouspromoter sequence for driving expression of the dsRNA in the plant,comprises a homogenic (prior to amplification) or heterogenic (secondarysiRNAs, following amplification) population of plant non-transcribabledsRNA. In embodiments according to the present disclosure, an antisenseRNA sequence may be a heterologous sequence.

The present methodology can be used for modulating gene expression suchas in a plant, the method comprising: (a) contacting a seed of the plantwith a dsRNA, under conditions which allow penetration of the dsRNA intothe seed, thereby introducing the dsRNA into the seed; and optionally(b) generating a plant of the seed.

When used for down-regulating a plant gene, the dsRNA is designed of thedesired specificity using bioinformatic tools which are well known inthe art (e.g., BLAST).

This methodology can be used in various applications starting from basicresearch such as in order to asses gene function and lasting ingenerating plants with altered traits which have valuable commercialuse.

Such plants can exhibit agricultural beneficial traits including alteredmorphology, altered flowering, altered tolerance to stress (i.e., bioticand/or abiotic), altered biomass vigor and/or yield and the like.

The phrase “abiotic stress” as used herein refers to any adverse effecton metabolism, growth, viability and/or reproduction of a plant. Abioticstress can be induced by any of suboptimal environmental growthconditions such as, for example, water deficit or drought, flooding,freezing, low or high temperature, strong winds, heavy metal toxicity,anaerobiosis, high or low nutrient levels (e.g. nutrient deficiency),high or low salt levels (e.g. salinity), atmospheric pollution, high orlow light intensities (e.g. insufficient light) or UV irradiation.Abiotic stress may be a short term effect (e.g. acute effect, e.g.lasting for about a week) or alternatively may be persistent (e.g.chronic effect, e.g. lasting for example 10 days or more). The presentdisclosure contemplates situations in which there is a single abioticstress condition or alternatively situations in which two or moreabiotic stresses occur.

According to one embodiment the abiotic stress refers to salinity.

According to another embodiment the abiotic stress refers to drought.

According to another embodiment the abiotic stress refers to atemperature stress.

As used herein the phrase “abiotic stress tolerance” refers to theability of a plant toendure an abiotic stress without exhibitingsubstantial physiological or physical damage (e.g. alteration inmetabolism, growth, viability and/or reproducibility of the plant).

As used herein the phrase “nitrogen use efficiency (NUE)” refers to ameasure of crop production per unit of nitrogen fertilizer input.Fertilizer use efficiency (FUE) is a measure of NUE. Crop production canbe measured by biomass, vigor or yield. The plant's nitrogen useefficiency is typically a result of an alteration in at least one of theuptake, spread, absorbance, accumulation, relocation (within the plant)and use of nitrogen absorbed by the plant. Improved NUE is with respectto that of a non-transgenic plant (i.e., lacking the transgene of thetransgenic plant) of the same species and of the same developmentalstage and grown under the same conditions.

As used herein the phrase “nitrogen-limiting conditions” refers togrowth conditions which include a level (e.g., concentration) ofnitrogen (e.g., ammonium or nitrate) applied which is below the levelneeded for optimal plant metabolism, growth, reproduction and/orviability.

As used herein the term/phrase “biomass”, “biomass of a plant” or “plantbiomass” refers to the amount (e.g., measured in grams of air-drytissue) of a tissue produced from the plant in a growing season. Anincrease in plant biomass can be in the whole plant or in parts thereofsuch as aboveground (e.g. harvestable) parts, vegetative biomass, rootsand/or seeds or contents thereof (e.g., oil, starch etc.).

As used herein the term/phrase “vigor”, “vigor of a plant” or “plantvigor” refers to the amount (e.g., measured by weight) of tissueproduced by the plant in a given time. Increased vigor could determineor affect the plant yield or the yield per growing time or growing area.In addition, early vigor (e.g. seed and/or seedling) results in improvedfield stand.

As used herein the term/phrase “yield”, “yield of a plant” or “plantyield” refers to the amount (e.g., as determined by weight or size) orquantity (e.g., numbers) of tissues or organs produced per plant or pergrowing season. Increased yield of a plant can affect the economicbenefit one can obtain from the plant in a certain growing area and/orgrowing time.

According to one embodiment the yield is measured by cellulose content,oil content, starch content and the like.

According to another embodiment the yield is measured by oil content.

According to another embodiment the yield is measured by proteincontent.

According to another embodiment, the yield is measured by seed number,seed weight, fruit number or fruit weight per plant or part thereof(e.g., kernel, bean).

A plant yield can be affected by various parameters including, but notlimited to, plant biomass; plant vigor; plant growth rate; seed yield;seed or grain quantity; seed or grain quality; oil yield; content ofoil, starch and/or protein in harvested organs (e.g., seeds orvegetative parts of the plant); number of flowers (e.g. florets) perpanicle (e.g. expressed as a ratio of number of filled seeds over numberof primary panicles); harvest index; number of plants grown per area;number and size of harvested organs per plant and per area; number ofplants per growing area (e.g. density); number of harvested organs infield; total leaf area; carbon assimilation and carbon partitioning(e.g. the distribution/allocation of carbon within the plant);resistance to shade; number of harvestable organs (e.g. seeds), seedsper pod, weight per seed; and modified architecture [such as increasestalk diameter, thickness or improvement of physical properties (e.g.elasticity)].

Improved plant NUE is translated in the field into either harvestingsimilar quantities of yield, while implementing less fertilizers, orincreased yields gained by implementing the same levels of fertilizers.Thus, improved NUE or FUE has a direct effect on plant yield in thefield.

As used herein “biotic stress” refers stress that occurs as a result ofdamage done to plants by other living organisms, such as bacteria,viruses, fungi, parasites, beneficial and harmful insects, weeds, andcultivated or native plants. Example 7 of the Examples section whichfollows, implements the present teachings towards conferring resistanceto Spodoptera littoralis.

As used herein the term “improving” or “increasing” refers to at leastabout 2%, at least about 3%, at least about 4%, at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90% or greater increase in NUE,in tolerance to stress, in yield, in biomass or in vigor of a plant, ascompared to a native or wild-type plants (i.e., isogenic plants (notmodified to comprise the dsRNA)) of the disclosure.

As mentioned the target gene of the dsRNA may not be an endogenous plantgene but rather a gene exogenous to the plant such as of aphytopathogenic organism which feeds on the plant or depends thereon forgrowth/replication (e.g., bacteria or viruses) and survival.

As used herein. The term “phytopathogen” refers to an organism thatbenefits from an interaction with a plant, and has a negative effect onthat plant.

Thus, according to an embodiment of the disclosure there is provided amethod of inhibiting expression of a target gene in a phytopathogenicorganism, the method comprising providing (e.g., feeding or contactingunder infecting conditions) to the phytopathogenic organism the plant asdescribed herein (at least part thereof includes the dsRNA), therebyinhibiting expression of a target gene in the phytopathogenic organism.

The phytopathogenic organism refers to a multicellular organism e.g.,insects, fungi, animals or a microorganism that can cause plant disease,including viruses, bacteria, fungi as well as oomycetes, chytrids,algae, and nematodes.

Reference herein to a “nematode” refers to a member of the phylumNematoda. Members of the family Heteroderidae are sedentary parasitesthat form elaborate permanent associations with the target hostorganism. They deprive nutrients from cells of an infected organismthrough a specialized stylet. The cyst nematodes (genera Heterodera andGlobodera) and root-knot nematodes (genus Meliodogyne), in particular,cause significant economic loss in plants, especially crop plants.Examples of cyst nematodes include, inter alia, H. avenae (cereal cystnematodes), H. glycines (beet cyst nematode) and G. pallida (potato cystnematode). Root-knot nematodes include, for example, M. javanica, M.incognita and M. arenaria. These pathogens establish “feeding sites” inthe plant, by causing the morphological transformation of root cellsinto giant cells. Hence, nematode “infestation” or “infection” refers toinvasion of and feeding upon the tissues of the host plant. Othernematodes that cause significant damage include the lesion nematodessuch as Pratylenchus, particularly P. penetrans, which infects maize,rice and vegetables, P. brachyurus which infects pineapple and P.thornei which infects inter alia, wheat.

Insects that may cause damage and disease in plants belong to threecategories, according to their method of feeding: chewing, sucking andboring. Major damage is caused by chewing insects that eat plant tissue,such as leaves, flowers, buds and twigs. Examples from this large insectcategory include beetles and their larvae (grubs), web-worms, bagwormsand larvae of moths and sawflies (caterpillars). By comparison, suckinginsects insert their mouth parts into the tissues of leaves, twigs,branches, flowers or fruit and suck out the plant's juices. Typicalexamples of sucking insects include but are not limited to aphids, mealybugs, thrips and leafhoppers. Damage caused by these pests is oftenindicated by discoloration, drooping, wilting and general lack of vigorin the affected plant.

According to a specific embodiment, the phytopathogen is prodentia ofthe family Noctuidae e.g., Spodoptera littoralis.

Examples of significant bacterial plant pathogens include, but are notlimited to, Burkholderia, Proteobacteria (Xanthomonas spp. andPseudomonas spp, Pseudomonas syringae pv. tomato).

A number of virus genera are transmitted, both persistently andnon-persistently, by soil borne zoosporic protozoa. These protozoa arenot phytopathogenic themselves, but parasitic. Transmission of the virustakes place when they become associated with the plant roots. Examplesinclude Polymyxa graminis, which has been shown to transmit plant viraldiseases in cereal crops and Polymyxa betae which transmits Beetnecrotic yellow vein virus. Plasmodiophorids also create wounds in theplant's root through which other viruses can enter.

Specific examples of viruses which can be targeted according to thepresent teachings includes, but are not limited to:

(1) Tobacco mosaic virus (TMV, RNA virus) which infects plants,especially tobacco and other members of the family Solanaceae.

(2) Tomato spotted wilt virus (TSWV, RNA virus) which causes seriousdiseases of many economically important plants representing 35 plantfamilies, including dicots and monocots. This wide host range ofornamentals, vegetables, and field crops is unique among plant-infectingviruses. Belongs to tospoviruses in the Mediterranean area, affectvegetable crops, especially tomato, pepper and lettuce (Turina et al.,2012, Adv Virus Res 84; 403-437).

(3) Tomato yellow leaf curl virus (TYLCV) which is transmitted bywhitefly, mostly affects tomato plants. Geminiviruses (DNA viruses) inthe genus Begomovirus (including sweepoviruses and legumoviruses)—mostdevastating pathogens affecting a variety of cultivated crops, includingcassava, sweet potato, beans, tomato, cotton and grain legumes (Rey etal. 2012, Viruses 4; 1753-1791). Members include TYLCV above and tomatoleaf curl virus (ToLCV).

(4) Cucumber mosaic virus (CMV)—CMV has a wide range of hosts andattacks a great variety of vegetables, ornamentals, and other plants (asmany as 191 host species in 40 families). Among the most importantvegetables affected by cucumber mosaic are peppers (Capsicum annuum L.),cucurbits, tomatoes (Lycopersicon esculentum Mill.), and bananas (MusaL. spp.).

Other vegetable hosts include: cucumber, muskmelon, squash, tomato,spinach, celery, peppers, water cress, beet, sweet potato, turnip,chayote, gherkin, watermelon, pumpkin, citron, gourd, lima bean, broadbean, onion, ground-cherry, eggplant, potato, rhubarb, carrot, dill,fennel, parsnip, parsley, luffa, and artichoke (Chabbouh and Cherif,1990, FAO Plant Prot. Bull. 38:52-53.).

Ornamental hosts include: China aster, chrysanthemum, delphinium,salvia, geranium, gilia, gladiolus, heliotrope, hyacinth, larkspur,lily, marigold, morning glory, nasturtium, periwinkle, petunia, phlox,snapdragon, tulip, and zinnia (Chupp and Sherf, 1960; Agrios, 1978).

(5) Potato virus Y (PVY)—one of the most important plant virusesaffecting potato production.

(6) Cauliflower mosaic virus (CaMV, DNA virus (Rothnie et al., 1994)).

(7) African cassava mosaic virus (ACMV).

(8) Plum pox virus (PPV) is the most devastating viral disease of stonefruit from the genus Prunus.

(9) Brome mosaic virus (BMV)—commonly infects Bromus inermis and othergrasses, can be found almost anywhere wheat is grown.

(10) Potato virus X (PVX) There are no insect or fungal vectors for thisvirus. This virus causes mild or no symptoms in most potato varieties,but when Potato virus Y is present, synergy between these two virusescauses severe symptoms in potato.

Additional Viruses:

Citrus tristeza virus (CTV)—causes the most economically damagingdisease to Citrus, including sour orange (Citrus aurantium), and anyCitrus species grafted onto sour orange root stock, sweet orange (C.sinensis), grapefruit (C. paradisi), lime and Seville orange (C.aurantifolia), and mandarin (C. reticulata). CTV is also known to infectAeglopsis chevalieri, Afraegle paniculata, Pamburus missionis, andPassiflora gracilis. CTV is distributed worldwide and can be foundwherever citrus trees grow.

Barley yellow dwarf virus (BYDV)—most widely distributed viral diseaseof cereals. It affects the economically important crop species barley,oats, wheat, maize, triticale and rice.

Potato leafroll virus (PLRV) infects potatoes and other members of thefamily Solanaceae.

Tomato bushy stunt virus (TBSV), RNA virus, a member of the genusTombusvirus and mostly affects tomatoes and eggplant.

Additional Reviews:

Hamilton et al., 1981, J Gen Virol 54; 223-241—mentions TMV, PVX, PVY,CMV, CaMV

Additional Scientific Papers:

Makkouk et al., 2012, Adv Virus Res 84; 367-402—Viruses affecting peasand beans with narrow (Faba bean necrotic yellow virus (FBNYN)) and wide(alfalfa mosaic virus (AMV) and CMV) host range.

Insect pests causing plant disease include those from the families of,for example, Apidae, Curculionidae, Scarabaeidae, Tephritidae,Tortricidae, amongst others.

The target gene of the phytopathogenic organism encodes a productessential to the viability and/or infectivity of the pathogen, thereforeits down-regulation (by the dsRNA) results in a reduced capability ofthe pathogen to survive and infect host cells. Hence, suchdown-regulation results in a “deleterious effect” on the maintenanceviability and/or infectivity of the phytopathogen, in that it preventsor reduces the pathogen's ability to feed off and survive on nutrientsderived from host cells. By virtue of this reduction in thephytopathogen's viability and/or infectivity, resistance and/or enhancedtolerance to infection by a pathogen is facilitated in the cells of theplant. Genes in the pathogen may be targeted at the mature (adult),immature (juvenile) or embryo stages.

Examples of genes essential to the viability and/or infectivity of thepathogen are provided herein. Such genes may include genes involved indevelopment and reproduction, e.g. transcription factors (see, e.g. Xueet al., 1993; Finney et al., 1988), cell cycle regulators such as wee-1and ncc-1 proteins (see, e.g. Wilson et al., 1999; Boxem et al., 1999)and embryo-lethal mutants (see, e.g. Schnabel et al., 1991); proteinsrequired for modeling such as collagen, ChR3 and LRP-1 (see, e.g. Yochemet al., 1999; Kostrouchova et al., 1998; Ray et al., 1989); genesencoding proteins involved in the motility/nervous system, e.g.acetycholinesterase (see, e.g. Piotee et al., 1999; Talesa et al., 1995;Arpagaus et al., 1998), ryanodine receptor such as unc-68 (see, e.g.Maryon et al., 1998; Maryon et al., 1996) and glutamate-gated chloridechannels or the avermeetin receptor (see, e.g., Cully et al., 1994;Vassilatis et al., 1997; Dent et al., 1997); hydrolytic enzymes requiredfor deriving nutrition from the host, e.g. serine proteinases such asHGSP-1 and HGSP-III (see, e.g. Lilley et al., 1997); parasitic genesencoding proteins required for invasion and establishment of the feedingsite, e.g. cellulases (see, e.g. de Boer et al., 1999; Rosso et al.,1999) and genes encoding proteins that direct production of stylar oramphidial secretions such as sec-1 protein (see, e.g. Ray et al., 1994;Ding et al., 1998); genes encoding proteins required for sex or femaledetermination, e.g. tra-1, tra-2 and egl-1, a suppressor of ced9 (see,e.g. Hodgkin, 1980; Hodgkin, 1977; Hodgkin, 1999; Gumienny et al., 1999;Zarkower et al., 1992); and genes encoding proteins required formaintenance of normal metabolic function and homeostasis, e.g. sterolmetabolism, embryo lethal mutants (see, e.g. Schnabel et al., 1991) andtrans-spliced leader sequences (see, e.g. Ferguson et al, 1996), pos-1,cytoplasmic Zn finger protein; pie-1, cytoplasmic Zn finger protein;mei-1, ATPase; dif-1, mitochondrial energy transfer protein; rba-2,chromatin assembly factor; skn-1, transcription factor; plk-1, kinase;gpb-1, G-protein B subunit; par-1, kinase; bir-1, inhibitor ofapoptosis; mex-3, RNA-binding protein, unc-37, G-protein B subunit;hlh-2, transcription factor; par-2, dnc-1, dynactin; par-6, dhc-1,dynein heavy chain; and pal-1, homeobox. Such genes have been clonedfrom parasitic nematodes such as Meliodogyne and Heterodera species orcan be identified by one of skill in the art using sequence informationfrom cloned C. elegans orthologs (the genome of C. elegans has beensequenced and is available, see The C. elegans Sequencing Consortium(1998)).

As used herein, a “pathogen resistance” trait is a characteristic of aplant that causes the plant host to be resistant to attack from apathogen that typically is capable of inflicting damage or loss to theplant. Once the phytopathogen is provided with the plant materialcomprising the dsRNA, expression of the gene within the target pathogenis suppressed by the dsRNA, and the suppression of expression of thegene in the target pathogen results in the plant being resistant to thepathogen.

In this case, the target gene can encode an essential protein ortranscribe an non-coding RNA which, the predicted function is forexample selected from the group consisting of ion regulation andtransport, enzyme synthesis, maintenance of cell membrane potential,amino acid biosynthesis, amino acid degradation, development anddifferentiation, infection, penetration, development of appressoria orhaustoria, mycelial growth, melanin synthesis, toxin synthesis,siderophore synthesis, sporulation, fruiting body synthesis, celldivision, energy metabolism, respiration, and apoptosis, among others.

According to a specific embodiment, the phytopathogenic organism isselected from the group consisting of a fungus, a nematode, a virus, abacteria and an insect.

To substantiate the anti-pest activity, the present teachings alsocontemplate observing death or growth inhibition and the degree of hostsymptomatology following said providing.

To improve the anti-phytopathogen activity, embodiments of the presentdisclosure further provide a composition that contains two or moredifferent agents each toxic to the same plant pathogenic microorganism,at least one of which comprises a dsRNA described herein. In certainembodiments, the second agent can be an agent selected from the groupconsisting of inhibitors of metabolic enzymes involved in amino acid orcarbohydrate synthesis; inhibitors of cell division; cell wall synthesisinhibitors; inhibitors of DNA or RNA synthesis, gyrase inhibitors,tubulin assembly inhibitors, inhibitors of ATP synthesis; oxidativephosphorylation uncouplers; inhibitors of protein synthesis; MAP kinaseinhibitors; lipid synthesis or oxidation inhibitors; sterol synthesisinhibitors; and melanin synthesis inhibitors.

In addition, plants generated according to the teachings of the presentdisclosure or parts thereof can exhibit altered nutritional ortherapeutic efficacy and as such can be employed in the food or feed anddrug industries. Likewise, the plants generated according to theteachings of the present disclosure or parts thereof can exhibit alteredoil or cellulose content and as such can be implemented in theconstruction or oil industry.

The seeds of the present disclosure can be packed in a seed containingdevice which comprises a plurality of seeds at least some of which(e.g., 5%, 10% or more) containing an exogenous dsRNA, wherein the seedis devoid of a heterologous promoter for driving expression of thedsRNA.

The seed containing device can be a bag, a plastic bag, a paper bag, asoft shell container or a hard shell container.

Reagents of the present disclosure can be packed in a kit including thedsRNA, instructions for introducing the dsRNA into the seeds andoptionally a priming solution. According to one embodiment, the dsRNAand priming solution are comprised in separate containers.

Compositions of some embodiments of the disclosure may, if desired, bepresented in a pack or dispenser device which may contain one or moreunit dosage forms containing the active ingredient. The pack may, forexample, comprise metal or plastic foil, such as a blister pack. Thepack or dispenser device may be accompanied by instructions forintroduction to the seed.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this disclosure maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of thedisclosure. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals there between.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

It is appreciated that certain features of the disclosure, which are,for clarity, described in the context of separate embodiments, may alsobe provided in combination in a single embodiment. Conversely, variousfeatures of the disclosure, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the disclosure. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and embodiments of the present disclosure asdelineated hereinabove and as claimed in the claims section below findexperimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate some embodiments of the disclosure in anon limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present disclosure include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Example 1: Protocols for dsRNA Production and Seed Treatment

Generating dsRNA/siRNA Sequences

The dsRNA sequences were custom-created for each gene using in vitrotranscription of PCR products. Part of the mRNA, including either theORF, 3′ UTR or 5′ UTR for which dsRNA to be produced was PCR-amplifiedusing gene-specific primers, which contain the sequence of the T7promoter on either side. This product was used as a template for dsRNAproduction using commercial kits such as the MaxiScript dsRNA kit (LifeTechnologies) or T7 High Yield RNA Synthesis kit (NEB). Next, the sampleis treated with DNase Turbo at 37° C. for 15-30 min followed by phenoltreatment and nucleic acid precipitation. Next, one of two differentreactions is carried out: (1) dsRNA is ready to use, (2) processing ofthe dsRNA with Dicer (Shortcut RNase III (NEB)) to create smallinterfering RNAs (siRNA).

Either dsRNA or a combination of dsRNA and siRNA were used for seedtreatments as described below.

General Seed Treatment Protocol for Gene Silencing Using a dsRNA/siRNAMixture

Uncoated organic corn seeds were from variety “popcorn”, uncoatedorganic whole grain rice seeds, organic soybean and wheat seeds werepurchased from Nitsat Haduvdevan (Israel), Fresh tomato seeds wereretrieved from M82 tomato fruits, which are propagated in-house.Uncoated or fresh plant seeds were washed with double distilled water(DDW) prior to treatment for four hours. Next, seeds were dried at 30°C. for 10-16 hours. Following the drying step, seeds were treated with asolution containing the dsRNA formulation, which is made of dsRNA at afinal concentration of 40-150 μg/ml in 0.1 mM EDTA. Treatment wasperformed by gently shaking the seeds in the solution for 24 hours in adark growth chamber at 15° C. Finally, seeds were washed twice brieflyand planted on soil or dried for 0-30 hours and germinated at 25° C. ina dark growth chamber and planted in soil or planted directly in soil.Control seeds were treated in a similar way, with a formulation thatlacked the dsRNA or with non-specific dsRNA.

Example 2: Stability of the dsRNA in Seedlings of Rice, Tomato andSorghum

As an example for an exogenous gene that is not present/expressed inplants, the ORFs encoding the replicase and coat protein of CGMMV(accession number AF417242) were used to as targets for dsRNA treatmentof plant seeds using the protocol described in Example 1. Rice, tomatoand sorghum seeds were washed for 4 hours at 20° C., tomato and sorghumwere dried at 30° C. and rice at 20° C. for overnight. Seeds wereimmediately treated at 15° C. with 132.7 μg/ml dsRNA (finalconcentration) for 39 hours for rice, 93.8 μg/ml dsRNA (finalconcentration) for 48 hours for tomato, and 75 μg/ml dsRNA (finalconcentration) for 40 hours for sorghum.

Briefly, the virus-derived ORFs were amplified by PCR with specificallydesigned forward and reverse primers that contain the T7 sequence(5′-TAATACGACTCACTATAGGG-3′, SEQ ID NO: 1) at their 5′ (see Table 2,below). PCR products were purified from agarose gel and since they carryT7 promoters at both ends they were used as templates for T7-dependentin-vitro transcription, resulting in dsRNA product of the CGMMV genes.PCR on a housekeeping gene, tubulin, was used as a positive control(forward primer 5′-GGTGCTCTGAACGTGGATG-3′ (SEQ ID NO: 2), and reverseprimer 5′-CATCATCGCCATCCTCATTCTC-3′(SEQ ID NO: 3)).

TABLE 2PCR primers served as Templates for in vitro Transcription and detection ofCGMMV, and CGMMV dsRNA products. Forward Reverse Virus Productprimer/SEQ ID primer/SEQ Name Name Product Sequence/SEQ ID NO: NO:ID NO: 1) CGMMV CGMVV TAATACGACTCACTATAGGGGGTAAGCG TAATACGACT Set 1:(NCBI dsRNA GCATTCTAAACCTCCAAATCGGAGGTTGG CACTATAGGG TAATACGA Accessionproduct 1 ACTCTGCTTCTGAAGAGTCCAGTTCTGTT GGTAAGCGGC CTCACTATA numberTCTTTTGAAGATGGCTTACAATCCGATCA ATTCTAAACC/ GGGGAAGA AF417242)CACCTAGCAAACTTATTGCGTTTAGTGCT (SEQ ID NO: 5) CCCTCGAATCTTATGTTCCCGTCAGGACTTTACTTAAT CTTCTTATGT ACTAAGC/TTTCTAGTTGCTTCACAAGGTACCGCTTTC TCCCGTCAGG/ (SEQ ID NO: 4)CAGACTCAAGCGGGAAGAGATTCTTTCCG (SEQ ID NO: 7) Set 2:CGAGTCCCTGTCTGCGTTACCCTCGTCTG ACTCAGCA TCGTAGATATTAATTCTAGATTCCCAGATGTCGTAGG GCGGGTTTTTACGCTTTCCTCAACGGTCC ATTG/(SEQTGTGTTGAGGCCTATCTTCGTTTCGCTTCT ID NO: 6) CAGCTCCACGGATACGCGTAATAGGGTCATTGAGGTTGTAGATCCTAGCAATCCTAC GACTGCTGAGTCGCTTAACGCCGTAAAGCGTACTGATGACGCGTCTACGGCCGCTAGG GCTGAGATAGATAATTTAATAGAGTCTATTTCTAAGGGTTTTGATGTTTACGATAGGG CTTCATTTGAAGCCGCGTTTTCGGTAGTCTGGTCAGAGGCTACCACCTCGAAAGCTTA GTTTCGAGGGTCTTCCCCTATAGTGAGTCGTATTA/(SEQ ID NO: 8) CGMVV TAATACGACTCACTATAGGGGCTTTACCG TAATACGACTSet 3: dsRNA CCACTAAGAACTCTGTACACTCCCTTGCG CACTATAGGG TAATACGA product 2GGTGGTCTGAGGCTTCTTGAATTGGAATA GCTTTACCGC CTCACTATATATGATGATGCAAGTGCCCTACGGCTCAC CACTAAGAAC/ GGGCATCACTTGTTATGACATCGGCGGTAACTATACG (SEQ ID CCATCGACCAGCACTTGTTCAAAGGTAGATCATATGT NO: 10) CCTAAAC/GCATTGCTGCAATCCGTGCCTAGATCTTA (SEQ ID AAGATGTTGCGAGGAATGTGATGTACAANO: 9) CGATATGATCACGCAACATGTACAGAGG CACAAGGGATCTGGCGGGTGCAGACCTCTTCCAACTTTCCAGATAGATGCATTCAGG AGGTACGATAGTTCTCCCTGTGCGGTCACCTGTTCAGACGTTTTCCAAGAGTGTTCCT ATGATTTTGGGAGTGGTAGGGATAATCATGCAGTCTCGTTGCATTCAATCTACGATAT CCCTTATTCTTCGATCGGACCTGCTCTTCATAGGAAAAATGTGCGAGTTTGTTATGCAG CCTTTCATTTCTCGGAGGCATTGCTTTTAGGTTCGCCTGTAGGTAATTTAAATAGTATT GGGGCTCAGTTTAGGGTCGATGGTGATGCCCTATAGTGAGTCGTATTA/(SEQ ID NO: 11)

dsRNA homologous to green mottle mosaic virus was observed to be stablein rice seedlings. Rice seeds were treated at 15° C. with 132.7 μg/mldsRNA (final concentration) for 39 hours and dsRNA was detected_by realtime polymerase chain reaction (RT-PCR) 1 week post germination.Detection of tubulin cDNA serves as a positive control for the cDNAquality. At two weeks post germination, dsRNA was detectable in 10 outof 10 seedlings. At 3 weeks post germination, dsRNA homologous to greenmottle mosaic virus was detected in 5 out of 5 samples of riceseedlings.

Tomato seeds were treated at 15° C. with 93.8 μg/ml dsRNA (finalconcentration) for 48 hours and sorghum seeds treated at 5 μg/ml dsRNA(final concentration) for 40 hours. CGMMV dsRNA was detected by RT-PCRin 5 out of 13 tomato seedlings tested at 10 day post-germination and 3out of four sorghum seedlings 4 weeks after germination.

The exogenous dsRNA was found to be stable for at least three weeks inrice seedlings and at least 10 days in tomato seedlings and four weeksin Sorghum plants.

Example 3: The dsRNA is not Integrated into the Genome of Rice

Rice seeds were treated with an exogenous dsRNA as in Example 2. Plantswere germinated and grown for five weeks, DNA was extracted and PCRreactions were performed to demonstrate that the dsRNA did not integrateinto the Rice's genome. Two sets of primers that gave a positivereaction when checked on the RNA level were used, set 1 (see Table 2) ofprimers were the set of primers used to amplify the template (all thedsRNA sequence). Set 2 (see Table 3) are the primers that were used inthe PCR above. A Rice endogenous housekeeping gene (tubulin) was used asa positive control for the PCR reaction (see Table 3).

Three different DNA PCR reactions were carried out on dsRNA treated anduntreated plants. No amplified DNA corresponding to CGMMV was detectedin any treated or untreated plant.

TABLE 3 Tubulin Primers Used for PCR Amplification. Primer Name andPrimer Sequence/ Primer Direction (SEQ ID NO:) Length osa_TubA1_736FGGTGCTCTGAACGTGGATG (SEQ 19 ID NO: 12) osa_TubA1_1342RCATCATCGCCATCCTCATTCTC 22 (SEQ ID NO: 13)

Example 4: Exogenous dsRNA Molecules are Highly Stable in Solution anddo not Get Incorporated into the Genome of Treated Plants

Corn seeds were treated using the protocol described in Example 1, seedswere washed for 4 h at 20° C., dried at 30° C. overnight and immediatelytreated with 40 μg/ml dsRNA (final concentration) directed against the13-glucuronidase (GUS) reporter gene for 60 hours at 15° C., dried andwere germinated. Leaves and roots were harvested from control anddsGUS-treated plants 7 and 15 days following germination. RNA wasextracted from the harvested tissues and RT-PCR with specific GUSprimers was run (Table 4). In addition, a corn endogenous housekeepinggene (ubiquitin) was used as a positive control for the PCR reaction.The GUS dsRNA molecules were found to be extremely stable in the treatedseeds, and can be detected in corn plants 7 and 15 days post germinationof the seeds.

GUS dsRNA was detected in corn seedlings by RT-PCR at 7 and 15 daysafter germination according to an aspect of the present disclosure. Atone week, GUS dsRNA was detected in shoots of nine of eleven cornseedlings tested. GUS dsRNA was not detected in untreated plants. At 1week post-germination, GUS dsRNA was detected in five of five cornseedlings' roots. At 15 days post germination, GUS dsRNA was detected incorn seedlings' roots. GUS dsRNA molecules do not get incorporated inthe genome of treated corn plants one week after germination asdetermined by agarose gel electrophoresis of DNA PCR reactions using GUSprimers on DNA isolated from treated corn plants.

TABLE 4 Primers for PCR Amplification of GUS and Ubiquitin Genes and GUSdsRNA product. Primer Primer Length Primer Sequence/SEQ ID NO: NameGUS T7_For TAATACGACTCACTATAGGGAGATCGACGGCCTGTGGGCATT C /(SEQ ID NO: 15)GUS T7_Rev TAATACGACTCACTATAGGGAGCATTCCCGGCGGGATAGTCT 43G /(SEQ ID NO: 16) GUS208For CAGCGCGAAGTCTTTATACC/(SEQ ID NO: 17) 43GUS289Rev CTTTGCCGTAATGAGTGACC/(SEQ ID NO: 18) 20 zmaUBQ-947FCCATAACCCTGGAGGTTGAG/(SEQ ID NO: 19) 20 zmaUBQ1043RATCAGACGCTGCTGGTCTGG/(SEQ ID NO: 20) 20 GUS dsRNATAATACGACTCACTATAGGGAGATCGACGGCCTGTGGGCATTC productAGTCTGGATCGCGAAAACTGTGGAATTGATCAGCGTTGGTGGGAAAGCGCGTTACAAGAAAGCCGGGCTATTGCTGTGCCAGGCAGTTTTAACGATCAGTTCGCCGATGCAGATATTCGTAATTATGCGGGCAACGTCTGGTATCAGCGCGAAGTCTTTATACCGAAAGGTTGGGCAGGCCAGCGTATCGTGCTGCGTTTCGATGCGGTCACTCATTACGGCAAAGTGTGGGTCAATAATCAGGAAGTGATGGAGCATCAGGGCGGCTATACGCCATTTGAAGCCGATGTCACGCCGTATGTTATTGCCGGGAAAAGTGTACGTATCACCGTTTGTGTGAACAACGAACTGAACTGGCAGACTATCCCGCCGGGAATGCTCCCTATAGTGAGTCGTATTA/(SEQ ID NO: 21)

Example 5: Fluorescence Microscopy of siRNA Sequences in Various PlantSeeds

Plant seeds as per the protocol described in example 1. Seeds werewashed for 4 h at 20° C., dried at 25° C. and were immediately treatedwith a fluorescent siRNA (siGLO, 2 μM final concentration, ThermoScientific) at 15° C. for 24 h. The quality of the siGLO beforeapplication to a plant seed was verified by gel electrophoresisanalysis. Bands corresponding to the expected size of 20-24 bp of thefluorescent siRNA molecules was detected.

Fluorescent pictures of the seeds were taken 24-48 hours post treatmentusing an Olympus microscope at the lowest objective magnification (5×for bigger seeds such as rice and tomato seeds, and 10× for smallerseeds such as Arabidopsis seeds). To eliminate the possibility ofnon-specific auto-fluorescence, dsRNA-treated seeds were compared tocontrol untreated seeds. Penetration of fluorescent siRNA molecules intoplant seeds was observed at 24 hours after seed treatment with siRNA at2 μM final concentration in rice seeds and tomato seeds.

Penetration of fluorescent siRNA molecules into rice seeds was observedat 24 hours following treatment with siGLO dsRNA.

In order to evaluate the distribution efficiency of the fluorescentsiRNA inside the seeds, different plant seeds were cut into slices andimaged with a fluorescent microscope 48 hours after treatment. Eachtreated seed was imaged alongside a control untreated seed. Light andfluorescent images were taken where applicable for rice, tomato,cucumber, bean, sorghum and wheat seed samples.

Penetration of fluorescent siRNA molecules into rice seeds was observedat 48 hours following treatment with siGLO dsRNA. siGLO-treated andcontrol rice seeds were sliced to view the interior distribution of thefluorescent dsRNA using a fluorescent microscope and fluorescent siRNAmolecules detected in the treated seed. Fluorescent siGLO RNA isdetected throughout the seed.

Penetration of fluorescent siRNA molecules into tomato seeds wasobserved at 48 hours following treatment with siGLO dsRNA. siGLO-treatedand control tomato seeds were sliced to view the interior distributionof the fluorescent dsRNA using a fluorescent microscope. FluorescentsiGLO RNA is detected in the endosperm and the embryo.

Penetration of fluorescent siRNA molecules into cucumber seeds wasobserved at 48 hours following treatment with siGLO dsRNA. siGLO-treatedand control cucumber seeds were sliced to view the interior distributionof the florescent dsRNA using a fluorescent microscope.

Penetration of fluorescent siRNA molecules into cucumber seeds wasobserved at Fluorescent siGLO RNA is detected in the endosperm and theembryo.

Penetration of fluorescent siRNA molecules is detected in sliced seedsof various plant species, including bean, tomato, sorghum and wheat, 48hours following treatment with siGLO dsRNA. siGLO-treated and controlseeds were sliced to view the interior distribution of the fluorescentdsRNA using a fluorescent microscope. Light images were also taken foreach seed and are shown alongside the fluorescent image of the seed forreference.

FIG. 1 presents fluorescent images of siGLO-treatment rice seeds over a24 hour period. The effect of incubation time with siGLO dsRNA onfluorescence intensity, indicating quantity and quality of dsRNApenetration, was tested. Control seeds that were left untreated (1),were imaged along with seeds treated with siGLO dsRNA for four differentincubation times; 10 min (2), 3.5 hours (3), 5.5 hours (4), and 24 hours(5).

It is clear that the siRNA is distributed at various levels between theembryo and the endosperm. Accordingly, dsRNA molecules enter the embryodirectly. Though not to be limited by any particular theory, the dsRNAmolecules are carried by the water-based solution used for the seedtreatment. The dsRNA molecules enter the endosperm as part of theendosperm's water-absorption process. These molecules then aretransferred to the embryo as it develops as part of the endosperm toembryo nutrient flow during germination and seed development.

These present findings suggest the RNA molecules used to treat the seedsboth penetrate the embryo and function in the embryo as it develops andalso penetrate the endosperm and feed the embryo following germination.

Example 6: Time Course Experiment with siGLO Treatment

A time course experiment was performed on rice seeds to monitor thekinetics of siGLO penetration into the seeds following the seedtreatment (FIG. 1). The results indicate that the siRNA efficientlypenetrates the plant seeds using the protocol described in Example 1.

Example 7: Example Embodiments of dsRNA Molecules

Example 7A provides A backbone sequence with two smRNA complementarysites and a helicase binding site:

(SEQ ID NO: 14) 5′ GCATCCTCATCTTAATCTCGGTGCTATCCTACCTGAGCTTGATATC TAGGCGAAGCAGCCCGAATGCTGCACCCTAGATGGCGAAAGTCCAGTAGCGATATCGAATTCCTCGAGGGATCCAAGCTTCCTTGTCTATCCCTCCTGAGCTGTTGATTTTATTCCATGT 3′.This example contains a sequence for mutated microRNA 390 binding(bold), followed by a helicase binding site (bold and underlined) and amicroRNA 390 binding sequence (underlined). DNA sequences forrestriction enzyme recognition are added for cloning of the sequence tobe silenced.

Example 7B is the same as example 7A, without the helicase binding site:

(SEQ ID NO: 22) GCATCCTCATCTTAATCTCGGTGCTATCCTACCTGAGCTTGATATCGATATCGAATTCCTCGAGGGATCCAAGCTTCCTTGTCTATCCCTCCTGAGCTGT TGATTTTATTCCATGT.

Example 7C provides a backbone sequence with two smRNA complementarysites and an helicase binding site:

(SEQ ID NO: 23) GCATCCTCATCTTAATCTCGTGATTTTTCTCTACAAGCGAAGATATC TAGGCGAAGCAGCCCGAATGCTGCACCCTAGATGGCGAAAGTCCAGTAGC GATATCGAATTCCTCGAGGGATCCAAGCTTTCTTGCTCAAATGAGTATTCCAG TTGATTTTATTCCATGT.Example 7C contains a sequence for microRNA 173 binding (bold), followedby a helicase binding site (bold and underlined) and thereverse-complement sequence of microRNA 828 binding sequence(underlined). DNA sequences for restriction enzyme recognition are addedfor cloning of the sequence to be silenced. In this case a singlecomplementary site is sufficient (i.e., miR173BS) yet a secondcomplementary site is placed on the complementary strand (i.e.,miR828BS) so as to enhance amplification from both strands.

Example 7D is the same as Example 7C, without the helicase binding site:

(SEQ ID NO: 24) GCATCCTCATCTTAATCTCGTGATTTTTCTCTACAAGCGAAGATATCGATATCGAATTCCTCGAGGGATCCAAGCTTTCTTGCTCAAATGAGTATTCCAG TTGATTTTATTCCATGT.

Example 8: Schematic Representation of the Solanum Lycopersicum TAS3Gene

FIG. 2A presents a schematic representation of the Solanum Lycopersicum(Lycopersicon esculentum) TAS3 gene. This gene contains two Mir390binding sites (BS). The 5′ Mir390BS has mutations in critical positionsfor Mir390 dependent cleavage and therefore it is bound by Mir390 butnot cleaved (will be referred hereafter as 5′ Mut Mir390BS). The 3′Mir390 does lead to Mir390 binding and cleavage. In between these twosequences there is a 234 bp sequence that contains all the differentta-siRNAs that will be created following RDRP recruitment, RNA dependentRNA polymerization and dicing (Allen et al. (2005). MicroRNA-DirectedPhasing during Trans-Acting siRNA Biogenesis in Plants. Cell, 121,207-221., Axtell et al. (2006). A Two-Hit Trigger for siRNA Biogenesisin Plants. Cell, 127, 565-577., Montgomery et al. (2008). Specificity ofARGONAUTE7-miR390 Interaction and Dual Functionality in TAS3Trans-Acting siRNA Formation. Cell, 133, 128-141).

Example 9: Additional dsRNA Constructs According to the PresentDisclosure

Example 9A provides dsRNA Construct #1 that is an exogenous triggercontrol. FIG. 2B presents a schematic representation of dsRNA Construct#1 that will serve as a control for the other experiments since itcontains only the exogenous sequence with no additional features thatshould lead to its amplification. The length of the exogenous sequenceis 234 bp, the same size of the original insert between the two Mir390BSin TAS3.

Example 9B provides dsRNA Construct #2 having a dual Mir390BS on sensestrand and an exogenous sequence. FIG. 2C presents schematicrepresentation of dsRNA Construct #2 having a dual Mir390BS on sensestrand and an exogenous sequence. Double-stranded RNA Construct #2 isbased on the dual Mir390BS from the TAS3 gene with an exogenous sequencereplacing the original insert between the two Mir390BS.

Example 9C provides dsRNA construct #3 having a Dual Mir390BS both onthe sense and antisense strands. FIG. 3 presents a schematic of dsRNAconstruct #3. This construct contains dual Mir390BS on both the senseand antisense strands and therefore we hypothesize that it willcontinuously recruit Mir390-Ago7 and RDRP to both strands. As a result,it is predicted to lead to long lasting amplification of the exogenoussequence and to ongoing production of its ta-siRNAs.

Example 9D provides dsRNA construct #4 having miR390BS as overhangs.FIG. 4 presents dsRNA construct #4 composed of two different strands.Having the Mir390BS present as overhangs will ease the initialrequirement for the unwinding of the dsRNA since the Mir390BS willalready be accessible for mir390 and Ago7 binding. The Mir390BSsequences will facilitate the unwinding and as a result thetranslocation into the processing center and the initiation of theentire process as explained in example #3 above.

Example 9E provides dsRNA construct #5 having Dual miR390BS sequencesand helicase binding sequences. FIG. 5 presents dsRNA construct #5. Thepresence of the helicase binding sequences will enable more efficientunwinding of the dsRNA through active recruitment of a helicase andtherefore leading to a strong and efficient amplification.

Example 9F provides dsRNA construct #6 having Mir390BS on both strandsand a helicase binding sequence (helicase BS) overhang. FIG. 6 presentsdsRNA construct #6 having Mir390BS on both strands and a helicaseoverhang. This dsRNA construct #6 is composed of two different strands.The sense strand is the same as in construct #3 and the antisense strandis the same as construct #3 with an addition of an overhang of ahelicase BS at the 3′ end. The helicase BS leads to recruitment of ahelicase that will unwind the dsRNA and enable efficient initiation ofthe entire process. Each of the strands will contain Mir390-Ago7sequences for binding and localization into the processing centerenabling long lasting amplification.

Example 9G provides dsRNA construct #7 having a sense dual Mir390BSsequence coupled with an antisense Mir4376BS. FIG. 7 presents dsRNAconstruct #7 containing a dualMir390BS on its sense strand and a singleMir4376BS on its antisense strand. The presence of ta-siRNA inducingmiRNAs on both strands will lead to ongoing amplification.

Example 9H provides dsRNA construct #8 having an Endogenous TriggerControl. FIG. 8 presents dsRNA construct #8 that is based on an exactendogenous insert sequence (the insert is the region between the twoMir390BS) in order to serve as an endogenous trigger control for thedsRNA construct #9 of Example 91.

Example 9I presents dsRNA construct #9 having a Mir390BS sequence andthe Endogenous insert of Example 9H. FIG. 9 presents a schematic ofdsRNA construct #9. Construct #9 maintains the endogenous sequence ofthe Mir390BS including the original insert region. This construct resultin production of ta-siRNAs targeting ARF3 and ARF4.

Example 10: Treatment of Seeds with ta-siRNA Constructs and Analysis ofRNA Levels

Tomato seeds were treated with dsRNA molecules corresponding to theconstructs and sequences of Table 5.

TABLE 5 dsRNA constructs SEQ Trigger Trigger ID # alias Sequence (5′-3′)Length DS/SS S/AS NO: 1 GFP234 CTAATACGACTCACTATAGGGAGATTTCCG 282 DSSense 320 TCCTCCTTGAAATCAATTCCCTTAAGCTCG ATCCTGTTGACGAGGGTGTCTCCCTCAAACTTGACTTCAGCACGTGTCTTGTAGTTCCCG TCGTCCTTGAAAGAGATGGTCCTCTCCTGCACGTATCCCTCAGGCATGGCGCTCTTGAAG AAGTCGTGCCGCTTCATATGATCTGGGTATCTTGAAAAGCATTGAACACCATAAGAGAA AGTAGTGACAAGTGTTGGCTCTCCCTATAGTGAGTCGTATTAG 2 GFP234Mir390 CTAATACGACTCACTATAGGGAGAGGTGC 325 DS Sense321 TATCCTACCTGAGCTTTTTCCGTCCTCCTTG AAATCAATTCCCTTAAGCTCGATCCTGTTGACGAGGGTGTCTCCCTCAAACTTGACTTCA GCACGTGTCTTGTAGTTCCCGTCGTCCTTGAAAGAGATGGTCCTCTCCTGCACGTATCCC TCAGGCATGGCGCTCTTGAAGAAGTCGTGCCGCTTCATATGATCTGGGTATCTTGAAAAG CATTGAACACCATAAGAGAAAGTAGTGACAAGTGTTGGCCCTTGTCTATCCCTCCTGAG CTTCTCCCTATAGTGAGTCGTATTAG 3GFP234Mir390X2 CTAATACGACTCACTATAGGGAGAAGCTC 368 DS Sense 322AGGAGGGATAGACAAGGGGTGCTATCCTA CCTGAGCTTTTTCCGTCCTCCTTGAAATCAATTCCCTTAAGCTCGATCCTGTTGACGAGG GTGTCTCCCTCAAACTTGACTTCAGCACGTGTCTTGTAGTTCCCGTCGTCCTTGAAAGAG ATGGTCCTCTCCTGCACGTATCCCTCAGGCATGGCGCTCTTGAAGAAGTCGTGCCGCTTC ATATGATCTGGGTATCTTGAAAAGCATTGAACACCATAAGAGAAAGTAGTGACAAGTGT TGGCAAGCTCAGGTAGGATAGCACCCCTTGTCTATCCCTCCTGAGCTTCTCCCTATAGT GAGTCGTATTAG 4 GFP234Mir390_CTAATACGACTCACTATAGGGAGAGGTGC 301 SS Sense 323 SenseTATCCTACCTGAGCTTTTTCCGTCCTCCTTG AAATCAATTCCCTTAAGCTCGATCCTGTTGACGAGGGTGTCTCCCTCAAACTTGACTTCA GCACGTGTCTTGTAGTTCCCGTCGTCCTTGAAAGAGATGGTCCTCTCCTGCACGTATCCC TCAGGCATGGCGCTCTTGAAGAAGTCGTGCCGCTTCATATGATCTGGGTATCTTGAAAAG CATTGAACACCATAAGAGAAAGTAGTGACAAGTGTTGGCCCTTGTCTATCCCTCCTGAG CT 5 GFP234Mir390_CTAATACGACTCACTATAGGGAGAGCCAA 258 SS Antisense 324 AntisenseCACTTGTCACTACTTTCTCTTATGGTGTTCA ATGCTTTTCAAGATACCCAGATCATATGAAGCGGCACGACTTCTTCAAGAGCGCCATGCC TGAGGGATACGTGCAGGAGAGGACCATCTCTTTCAAGGACGACGGGAACTACAAGACA CGTGCTGAAGTCAAGTTTGAGGGAGACACCCTCGTCAACAGGATCGAGCTTAAGGGAA 6 GFP234Mir390_CTAATACGACTCACTATAGGGAGAGGTGC 375 DS Sense 325 HelicaseTATCCTACCTGAGCTTTTTCCGTCCTCCTTG AAATCAATTCCCTTAAGCTCGATCCTGTTGACGAGGGTGTCTCCCTCAAACTTGACTTCA GCACGTGTCTTGTAGTTCCCGTCGTCCTTGAAAGAGATGGTCCTCTCCTGCACGTATCCC TCAGGCATGGCGCTCTTGAAGAAGTCGTGCCGCTTCATATGATCTGGGTATCTTGAAAAG CATTGAACACCATAAGAGAAAGTAGTGACAAGTGTTGGCGCTACTGGACTTTCGCCATC TAGGGTGCAGCATTCGGGCTGCTTCGCCTACCTTGTCTATCCCTCCTGAGCTTCTCCCTAT AGTGAGTCGTATTAG 7 GFP234Mir390_CTAATACGACTCACTATAGGGAGAAGCTC 344 SS Sense 326 Helicase_AGGAGGGATAGACAAGGGGTGCTATCCTA Sense CCTGAGCTTTTTCCGTCCTCCTTGAAATCAATTCCCTTAAGCTCGATCCTGTTGACGAGG GTGTCTCCCTCAAACTTGACTTCAGCACGTGTCTTGTAGTTCCCGTCGTCCTTGAAAGAG ATGGTCCTCTCCTGCACGTATCCCTCAGGCATGGCGCTCTTGAAGAAGTCGTGCCGCTTC ATATGATCTGGGTATCTTGAAAAGCATTGAACACCATAAGAGAAAGTAGTGACAAGTGT TGGCAAGCTCAGGTAGGATAGCACCCCTTGTCTATCCCTCCTGAGCT 8 GFP234Mir390_ CTAATACGACTCACTATAGGGAGAAGCTC 394 SSAntisense 327 Helicase_ AGGAGGGATAGACAAGGGGTGCTATCCTA AntiSenseCCTGAGCTTGCCAACACTTGTCACTACTTT CTCTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAGCGGCACGACTTCTT CAAGAGCGCCATGCCTGAGGGATACGTGCAGGAGAGGACCATCTCTTTCAAGGACGAC GGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAGGGAGACACCCTCGTCAACAGGA TCGAGCTTAAGGGAATTGATTTCAAGGAGGACGGAAAAAGCTCAGGTAGGATAGCACC CCTTGTCTATCCCTCCTGAGCTTAGGCGAAGCAGCCCGAATGCTGCACCCTAGATGGCG AAAGTCCAGTAGC 9 GFP234Mir390_CTAATACGACTCACTATAGGGAGAGGTGC 347 DS Sense 328 Mir4376TATCCTACCTGAGCTTTTTCCGTCCTCCTTG AAATCAATTCCCTTAAGCTCGATCCTGTTGACGAGGGTGTCTCCCTCAAACTTGACTTCA GCACGTGTCTTGTAGTTCCCGTCGTCCTTGAAAGAGATGGTCCTCTCCTGCACGTATCCC TCAGGCATGGCGCTCTTGAAGAAGTCGTGCCGCTTCATATGATCTGGGTATCTTGAAAAG CATTGAACACCATAAGAGAAAGTAGTGACAAGTGTTGGCTCGCAGGAGAGATGACACC AGACCTTGTCTATCCCTCCTGAGCTTCTCCCTATAGTGAGTCGTATTAG 10 TAS3 CTAATACGACTCACTATAGGGAGATTTCTC 282 DS Sense329 ACCGCTTTTTTTTTTCTGTTGTGTATTCTCT TTTTTGACTTGTTGCCTTTCGTTCCTCTACCTACCCCATTCTTCTTGACCTTGTAAGACCTT TTCTTGACCTTGTAAGACCCCGTGTTATCTCTTACGTCTTTATGTTTTGTTTTTTTGCAAA TCTTACGTCATGACTTCTTCATGTAAGCTTTGTTTGGTCTCCTTCTTCTTTCCTACTCAACT CTCGTTCTCCTTTCTCCCTATAGTGAGTCGT ATTAG 11TAS3Mir390 CTAATACGACTCACTATAGGGAGAGGTGC 325 DS Sense 330TATCCTACCTGAGCTTTTTCTCACCGCTTTT TTTTTTCTGTTGTGTATTCTCTTTTTTGACTTGTTGCCTTTCGTTCCTCTACCTACCCCATTC TTCTTGACCTTGTAAGACCTTTTCTTGACCTTGTAAGACCCCGTGTTATCTCTTACGTCTTT ATGTTTTGTTTTTTTGCAAATCTTACGTCATGACTTCTTCATGTAAGCTTTGTTTGGTCTCC TTCTTCTTTCCTACTCAACTCTCGTTCTCCTTCCTTGTCTATCCCTCCTGAGCTTCTCCCTA TAGTGAGTCGTATTAG 12 GUS234GCCACTTGCAAAGTCCCGCTAGTGCCT 236 DS Sense 331 TGTCCAGTTGCAACCACCTGTTGATCCGCATCACGCAGTTCAACGCTGACATCA CCATTGGCCACCACCTGCCAGTCAACAGACGCGTGGTTACAGTCTTGCGCGACA TGCGTCACCACGGTGATATCGTCCACCCAGGTGTTCGGCGTGGTGTAGAGCATT ACGCTGCGATGGATTCCGGCATAGTTAAAGAAATCATGGAAGTAAGC 13 GUS234Mir390 GGTGCTATCCTACCTGAGCTTCCACTT 278 DSSense 332 GCAAAGTCCCGCTAGTGCCTTGTCCAG TTGCAACCACCTGTTGATCCGCATCACGCAGTTCAACGCTGACATCACCATTGG CCACCACCTGCCAGTCAACAGACGCGTGGTTACAGTCTTGCGCGACATGCGTCA CCACGGTGATATCGTCCACCCAGGTGTTCGGCGTGGTGTAGAGCATTACGCTGC GATGGATTCCGGCATAGTTAAAGAAATCATGGAAGTAAGCCTTGTCTATCCCTC CTGAGCTC

GFP234 (FIG. 10A; Trigger #1), GFP234Mir390 (Trigger #2), TAS3 (Trigger#10) and TAS3Mir390 (FIG. 10B, Trigger #11) were prepared as provided inExample 1. A final concentration of 50 μg/ml dsRNA diluted with 0.1 mMEDTA was used. Treatment was performed by gently shaking the seeds inthe solution for 24 hours in a dark growth chamber at 15° C. followed bywashing with water three times for one minute. After treatment, seedswere germinated either on wet paper or in soil and grown at about 25° C.with a 16 hour photoperiod. The plants germinated in soil were wateredwith tap water as necessary. Seeds that were treated with a similarsolution not containing dsRNA (e.g., 0.1 mM EDTA “EDTA”) were germinatedand grown alongside the treated plants as a control.

Total RNA was extracted from whole seedlings, leaves or roots ofgerminated seeds seven, 14 and 30 days post treatment. For seeds thatwere germinated on paper, the entire seedling was harvested after sevendays. For seeds that were germinated in soil, the leaves and roots wereharvested and analyzed separately 14 and 30 days after treatment.

cDNA was prepared using oligo-dT primers and the expression levels ofTAS3, ARF3 and ARF4 was determined by real-time PCR with SYBR Green(Quanta BioSciences). The results are presented in FIG. 11A-E. Thehouse-keeping genes Expressed and Tip41 were used as endogenous controlgenes to normalize for input RNA amounts. ARF3 and ARF4 genes areregulated by the TAS3 system and their expression is predicted todecrease following TAS3Mir390 treatment. No significant difference inTAS3, ARF3 or ARF4 expression was detected in seedlings when comparingthe TAS3 construct with the TAS3Mir390 construct seven days aftertreatment (t-test, p-value>0.05). Similarly, no significant differencein TAS3, ARF3 or ARF4 expression was detected in leaves when comparingthe two constructs 14 days after treatment. When comparing expressionlevels in roots 14 days after treatment, a down-regulation trend wasobserved for ARF3 and ARF4 genes following treatment with the TAS3Mir390construct (FIGS. 11A-B). FIG. 11A shows relative quantification of ARF3mRNA following treatment with either TAS3 Insert or Mir390BS TAS3 dsRNAconstructs. Each point represents the expression value per individualplant. Expression values were normalized to the average expressionvalues of all plants treated with TAS3_Insert, which was set to 100%.The red line represents the normalized average expression values foreach treatment. FIG. 11B shows the same analysis for ARF4 mRNA levels.

The RNA extracted from seven-day old seedlings, as well as RNA extractedfrom 30-day old roots and 30-day old leaves was used in a second cDNAreaction with random primers and then subjected to real-time PCR withSYBR Green (Quanta BioSciences). The primers used for real-time werederived from the GFP sequence that appears in the dsRNA constructs.Therefore, this analysis provides an indication for the presence of thedsRNA that was used for seed treatment and/or for RNA that wassynthesized from this dsRNA in the plant tissue. Expressed and Tip41were used as endogenous control genes. For seven day old seedlings, a2.6 fold difference in GFP level was observed in plants treated withGFP234 compared to plants treated with TAS3 (t-test, p-value=0.08).However, no significant difference in GFP level was observed whencomparing between GFP234 and GFP234Mir390 treatments (FIGS. 11C-E). FIG.11C shows normalized Ct values for all treatments analyzed; for each RNAsample tested, the Ct value obtained from the real-time amplificationplot was normalized to the average Ct value of the two endogenouscontrol genes. This value was then subtracted from the number 50 toassign larger values for higher expression levels. Each dot representsone plant and the red line represents the average value per treatment.RNA samples that gave no Ct values were assigned a value of 40. FIG. 11Dshows a comparison between GFP234 and TAS3 treatments. FIG. 11E shows acomparison between GFP234 and GFP234Mir390 treatments. For 30 day oldroots, no significant difference in GFP level was observed whencomparing between GFP234 and GFP234Mir390 treatments. For 30 day oldleaves, a significant (t-test, p-value=0.005), 7.1-fold difference inGFP level was observed in plants treated with GFP234Mir390 compared toplants treated with GFP234 (FIG. 11F).

Example 11: Detection of GFP Sequence in Plants Following Seed Treatmentwith ta-si dsRNA Constructs

In a second experiment, tomato seeds were treated with dsRNA moleculescorresponding to GFP234 (Trigger#1), GFP234Mir390 (Trigger#2),GFP234Mir390X2 (Trigger#3), GFP234Mir390_Helicase (Trigger#6),GFP234Mir390_Mir4376 (Trigger#9), TAS3 (Trigger#10) and TAS3Mir390(Trigger#11) as provided above in Example 10 and Table 5, according tothe protocol described in Example 10. A final concentration of 50 μg/mldsRNA diluted with 0.1 mM EDTA was used. Treatment was performed bygently shaking the seeds in the solution for 24 hours in a dark growthchamber at 15° C. followed by washing with water three times for oneminute. After treatment, seeds were germinated on wet paper and grown atabout 25° C. with 16 hours photoperiod. Seeds that were treated withEDTA solution alone were germinated and grown alongside the treatedplants as a control.

Total RNA was extracted from shoots (including hypocotyl, cotyledon andshoot apical meristem) seven and fourteen days after treatment. cDNA wasprepared using random primers and the presence of the GFP sequence wasdetermined and quantified by real-time PCR as described in Example 10. Asignificant difference in GFP level was observed in plants seven daysafter treatment with GFP234Mir390, GFP234Mir390_Helicase orGFP234Mir390_Mir4376 dsRNAs compared to treatment with GFP234,TAS3Mir390 or EDTA. The GFP level detected was between 5.9 fold(following treatment with GFP234Mir390 Mir4376) to 25 fold (followingtreatment with GFP234Mir390_Helicase) higher compared to plants treatedwith GFP234, with a p-value<0.05 (t-test). No significant difference wasdetected when comparing GFP234Mir390X2 to GFP234 treatment (FIGS.12A-E). The analyses were performed as described for FIGS. 11C-F ofExample 10. FIG. 12A shows normalized Ct values for all treatments. FIG.12B shows a comparison between GFP234 and GFP234Mir390 treatments. FIG.12C shows a comparison between GFP234 and GFP234Mir390_Helicasetreatments. FIG. 12D shows a comparison between GFP234 andGFP234Mir390_Mir4376treatments. FIG. 12E shows a comparison betweenGFP234 and GFP234Mir390X2 treatments.

A significant, 33 fold difference in GFP level was observed in plants 14days after treatment with GFP234Mir390X2 dsRNA compared to treatmentwith GFP234 (t-test, p-value<0.05). Higher levels of GFP were alsodetected for GFP234Mir390, GFP234Mir390 Helicase and GFP234Mir390Mir4376 treatments compared to GFP234 treatment, but with no significantdifference (FIGS. 13A-B). The analyses were performed as described forFIGS. 11C-F. FIG. 13A shows normalized Ct values for all treatments.FIG. 13B shows a comparison between GFP234 and GFP234Mir390X2treatments.

The same cDNA prepared from RNA extracted from seven-day old seedlings,was used in a second real-time PCR, where the expression levels of TAS3,ARF3 and ARF4 was determined as described in Example 10 (except thatrandom primers, and not oligo-dT primers were used in the cDNAreaction). No significant difference in TAS3, ARF3 or ARF4 expressionwas detected in seedlings when comparing between TAS3 and TAS3Mir390treatments (t-test, p-value>0.05).

Example 12: Detection of GFP Sequence in Plants Following Seed Treatmentwith ta-si dsRNA Constructs

Tomato seeds were treated with dsRNA molecules corresponding to GFP234(Trigger#1), GFP234Mir390_Helicase (Trigger#6) and GFP234Mir390_Mir4376(Trigger#9), as provided above in Example 10 and Table 4, according tothe protocol described in Example 1. A final concentration of 50 μg/mldsRNA diluted with 0.1 mM EDTA was used. Treatment was performed bygently shaking the seeds in the solution for 24 hours in a dark growthchamber at 15° C. followed by washing with water three times for oneminute. After treatment, seeds were germinated on wet paper and grown atabout 25° C. with 16 hours photoperiod. Seeds that were treated withEDTA solution alone were germinated and grown alongside the treatedplants as a control.

Total RNA was extracted from shoots (including hypocotyl, cotyledon andshoot apical meristem) seven days after treatment. cDNA was preparedusing random primers and the presence of the GFP sequence was determinedand quantified by real-time PCR as described in Example 10. Nosignificant difference in GFP levels was observed when comparing theGFP234 treated plants to the GFP234Mir390_Helicase orGFP234Mir390_Mir4376 treated plants (Dunnett's test).

Example 13: Detection of GFP Sequence in Plants Following Seed Treatmentwith ta-si dsRNA Constructs

Tomato seeds were treated with dsRNA molecules corresponding to GFP234(Trigger#1), GFP234Mir390 (Trigger#2), GFP234Mir390X2 (Trigger#3),GFP234Mir390 Helicase (Trigger#6), GFP234Mir390_Mir4376 (Trigger#9),TAS3 (Trigger#10) and TAS3Mir390 (Trigger#11) as provided above inExample 10 and Table 5, according to the protocol described inExample 1. A final concentration of 50 μg/ml dsRNA diluted with 0.1 mMEDTA was used. Treatment was performed by gently shaking the seeds inthe solution for 24 hours in a dark growth chamber at 15° C. followed bywashing with water three times for one minute. After treatment, seedswere germinated on wet paper and grown at about 25° C. with 16 hoursphotoperiod. Seeds that were treated with EDTA solution alone weregerminated and grown alongside the treated plants as a control.

Total RNA was extracted from shoots (including hypocotyl, cotyledon andshoot apical meristem) seven and 14 days after treatment. cDNA wasprepared using random primers and the presence of the GFP sequence wasdetermined and quantified by real-time PCR as described in Example 10.Seven days after treatment, a significant, 10-fold difference in GFPlevel was observed in plants treated with GFP234Mir390 compared toplants treated with GFP234 (Dunnett's test, p-value=0.035). A 6.6-folddifference in GFP level was observed in plants following treatment withGFP234Mir390_Helicase compared to treatment with GFP234 (Dunnett's test,p-value=0.11). For 14 days old plants, a significant, 34-fold differencein GFP level was observed in plants treated with GFP234Mir390_Helicasecompared to plants treated with GFP234 (Dunnett's test, p-value=0.0004).An 8.5-fold difference in GFP level was observed in plants followingtreatment with GFP234Mir390 compared to treatment with GFP234 (Dunnett'stest, p-value=0.058). FIG. 14A shows normalized Ct values seven daysafter treatment. FIG. 14B shows normalized Ct values 14 days aftertreatment. The analysis was performed essentially as described for FIG.11C-F, except that instead of subtracting the normalized Ct values fromthe number 50 to assign larger values for higher GFP levels, an inversey-axis is presented.

RNA extracted from seven-day old shoots treated with TAS3 (Trigger#10)and TAS3Mir390 (Trigger#11) dsRNAs was used in a second real-time PCR todetermine the expression levels of TAS3, ARF3 and ARF4 as described inExample 11. A significant, 1.3-fold up-regulation in ARF4 expression wasdetected in plants following treatment with TAS3Mir390 compared totreatment with TAS3 (Dunnett's test, p-value=0.05). No significantdifference in TAS3 or ARF3 expression was detected in those plants.

Example 14: Detection of Gus Sequence in Plants Following Seed Treatmentwith ta-si dsRNA Constructs

Tomato seeds were treated with dsRNA molecules corresponding to GUS234(Trigger#12) and GUS234Mir390 (Trigger#13), as provided above in Example10 and Table 5, according to the protocol described in Example 1. Afinal concentration of 50 μg/ml dsRNA diluted with 0.1 mM EDTA was used.Treatment was performed by gently shaking the seeds in the solution for24 hours in a dark growth chamber at 15° C. followed by washing withwater three times for one minute. After treatment, seeds were germinatedon wet paper and grown at about 25° C. with 16 hours photoperiod. Seedsthat were treated with EDTA solution alone were germinated and grownalongside the treated plants as a control.

Total RNA was extracted from shoots (including hypocotyl, cotyledon andshoot apical meristem) seven and 14 days after treatment. cDNA wasprepared using random primers and the presence of the GUS sequence wasdetermined and quantified by real-time PCR as described in Example 10. Asignificant increase in GUS levels was observed in plants seven daysafter treatment with GUS234Mir390 compared to treatment with GUS234(Dunnett's test, p-value=0.0005). Most of the RNA samples extracted fromGUS234-treated plants gave no Ct value, meaning GUS was not detected inthose samples. Accordingly, these samples were assigned a value of 40.The resulting difference in the average Ct value between the GUS234 andthe GUS234Mir390 treatments was calculated to be about 10, translatinginto a 994-fold difference in GUS levels. For 14 days old plants, a12.2-fold difference in GUS levels was observed (Dunnett's test,p-value=0.03865). FIG. 15A shows normalized Ct values seven days aftertreatment with the two dsRNA constructs. FIG. 15B shows normalized Ctvalues 14 days after treatment. The analysis was performed as describedfor FIGS. 14A-B.

Example 15: Small RNA Deep Sequencing of Plants Following Seed Treatmentwith ta-si dsRNA Constructs

RNA samples from the seven day old shoots described in Examples 11 and14 were further analyzed by small RNA deep sequencing. cDNA librarieswere prepared with an Illumina TruSeq™ Small RNA kit according to themanufacturer's protocol, and sequenced by Illumina MiSeq® instrument.Each cDNA library was prepared from RNA pooled from three plantsoriginating from the same treatment. For GFP-based dsRNAs, a total often libraries were prepared. Two libraries (representing a total of sixplants) were prepared from GFP234 treated plants, three libraries (nineplants) were prepared from GFP234Mir390 treated plants, one library(three plants) was prepared from GFP234Mir390X2 treated plants, twolibraries (six plants) were prepared from GFP234Mir390_Helicase treatedplants and two libraries (six plants) were prepared from TAS3Mir390treated plants. Table 6 shows the average Ct value of each of the pooledRNA samples, according to the real-time PCR analysis shown in FIG. 12A.The values presented in the table were normalized by subtracting theaverage Ct value of RNA pooled from the TAS3Mir390 treatment. Lowquality reads and reads that contain adaptor sequences were filtered outfrom the raw sequencing data. Table 6 summarizes the number of readsfrom each library that were mapped to GFP. In accordance with thereal-time PCR results, more reads were mapped to the GFP sequence in theGFP234Mir390 and GFP234Mir390_Helicase treatments compared to the GFP234treatment.

TABLE 6 Small RNA MiSeq analysis of RNA extracted from plants followingseed treatment with ta-si-GFP dsRNA constructs GFP234Mir TreatmentGFP234 GFP234Mir390 GFP234Mir390_Helicase 390X2 TAS3Mir390 RT-PCR 1.32.8 3.2 6.6 9.5 7.9 5.9 2.7 0.0 4.0 normalized Ct value Libraries size1.23 1.24 2.09 1.46 1.4 1.59 1.23 1 1.61 1.07 ratio Total # of reads 3612 74 130 273 122 117 20 0 2 mapped to GFP234 sequence Normalized # of29 10 35 89 195 77 95 20 0 2 reads mapped to GFP234 sequence

For GUS-based dsRNAs, two libraries were prepared, one library(representing a total of three plants) was prepared from GUS234 treatedplants and one library (three plants) was prepared from GUS234Mir390treated plants. Table 7 shows the average Ct value of each of the pooledRNA samples, according to the real-time PCR analysis shown in FIG. 15A.The values presented in the Table were normalized by subtracting theaverage Ct value of RNA pooled from the GUS234 treatment. Table 7summarizes the number of reads from each library that were mapped toGUS. Data was analyzed as described for Table 6. In accordance with thereal-time PCR results, no reads were mapped to the GUS sequence in theGUS234 treatment while some reads were mapped to GUS in the GUS234Mir390treatment.

TABLE 7 Small RNA MiSeq analysis of RNA extracted from plants followingseed treatment with ta-si-GUS dsRNA constructs. Treatment GUS234GUS234Mir390 RT-PCR 0 7.6 normalized Ct value Libraries size 1 1.77ratio Total # of reads 0 27 mapped to GUS234 Normalized # of 0 15 readsmapped to GUS234 sequence

Example 16: Additional dsRNA Constructs

Additional dsRNA constructs based on the constructs and sequencesprovided in Examples 9 and 10 are provided. Deep-Sequencing analysisdescribed in Example 15 indicated that the first nucleotide in the smallRNA reads mapped to the endogenous TAS3 transcript is predominantly “T”.The most abundant reads mapped to the TAS3 transcript were located atpositions 37, 38, 79, 100, 101, 103, 184 and 185 of the 234nt sequenceand with the exception of the reads mapped to position 185, where thefirst nucleotide was A, the first nucleotide was T. Therefore, theinclusion of a “T” or “A” in the sequence of the target gene of interestthat is flanked by the two miR390 binding sites is expected to improvethe efficiency of cleavage and direct it to specific sites within thesequence or alternatively improves the interaction of the resultingsmall RNAs with downstream effectors.

Trigger #14 (SEQ ID No. 314), is designed as a modified GFP234Mir390sequence (based on Example 9B and trigger #2), where the nucleotides atpositions 37, 38, 79, 100, 101, 103 and 184 are “T”, and the nucleotideat position 185 is A (only positions 37, 79 and 101 were changed.Sequence appears in 5′-3′ orientation, the mentioned positions are inlowercase, bold).

SEQ ID No. 314: GGTGCTATCCTACCTGAGCTTTTTCCGTCCTCCTTGAAATCAATTCCCTTAAGCTCGttCCTGTTGACGAGGGTGTCTCCCTCAAACTTGACTTCAGCAtGTGTCTTGTAGTTCCCGTCGttCtTGAAAGAGATGGTCCTCTCCTGCACGTATCCCTCAGGCATGGCGCTCTTGAAGAAGTCGTGCCGCTTCATATGATCTGGGtaTCTTGAAAAGCATTGAACACCATAAGAGAAAGTAGTGACAAGTGTTGGCCCTTGTCTATCCCTCCTGAGCT

Trigger #15 (SEQ ID No. 315) is designed as a modified TAS3Mir390sequence (based on Example 91 and trigger #11). In this sequence, four21 nucleotides segments that begin with “TT” are selected from theGFP234 sequence and used to replace the original 21 nucleotide segmentsfrom the TAS3Mir390 sequence at positions 37, 79, 100 and 184. Thesepositions are in phase with the miR390-guided cleavage site (sequenceappears in 5′-3′ orientation, the mentioned four segments are inlowercase, bold).

SEQ ID No. 315: GGTGCTATCCTACCTGAGCTTTTTCTCACCGCTTTTTTTTTTCTGTTGTGTATTCTCtttccgtcctccttgaaatcaGTTCCTCTACCTACCCCATTCttcccttaagctcgatcctgtttgacttcagcacgtgtcttgGTGTTATCTCTTACGTCTTTATGTTTTGTTTTTTTGCAAATCTTACGTCATGACTTCTTCATGttcccgtcgtccttgaaagagCTTCTTTCCTACTCAACTCTCGTTCTCCTTCCTTGTCTATCCCTCCTGAGCT

Trigger #16 (SEQ ID No. 316) is designed as a modified GFP234Mir390sequence (based on Example 9B and trigger #2), where “T” appears every21 nucleotides. The most 3′ “T” is located at position 226 of the GFP234sequence, 21 nucleotides upstream to the miR390-guided cleavage site andall other “T” positions are in phase with this site (sequence appears in5′-3′ orientation, only mutated nucleotides are in lowercase, bold).

SEQ ID No. 316: GGTGCTATCCTACCTGAGCTTTTTCCGTCCTCCTTGtAATCAATTCCCTTAAGCTCGtTCCTGTTGACGAGGGTGTCTtCCTCAAACTTGACTTCAGCAtGTGTCTTGTAGTTCCCGTCGTCCTTGAAAGAGATGGTCCTCTCCTGCACGTATCCCTCAGGCtTGGCGCTCTTGAAGAAGTCGTGCCGCTTCATATGATCTGGGTATCTTGAAAAGCATTGAACAtCATAAGAGAAAGTAGTGACAtGTGTTGGCCCTTGTCTATCCCTCCTGAGCT

Trigger #17 (SEQ ID No. 317) is designed as a modified GFP234Mir390sequence (based on Example 9B and trigger #2), where ten 21 nucleotidesegments that originate from the full-length GFP sequence and beginswith a “T” are placed in tandem to produce the 234 nucleotide sequence.The first 15 and the last 9 nucleotides are from the endogenous TAS3sequence, in order to position the ten segments in-phase with themiR390-guided cleavage site (sequence appears in 5′-3′ orientation, thefirst “T” in each segment is in lowercase, bold, and the TAS3 sequenceis underlined).

SEQ ID No. 317: GGTGCTATCCTACCTGAGCTT TTTCTCACCGCTTTT tAATGGTTGTCTGGTAAAAGGtCGCCAATTGGAGTATTTTGTtGATAATGATCAGCGAGTTGCtCTTCGATGTTGTGGCGGGTCtTGAAGTTGGCTTTGATGCCGtTCTTTTGCTTGTCGGCCATGtGTATACGTTGTGGGAGTTGTtTGTATTCCAACTTGTGGCCGtGTTTCCGTCCTCCTTGAAATtTCCCTTAAGCTCGATCCTGTG TTC TCCTTCCTTGTCTATCCCTCCTGAGCT

Tomato seeds are treated with dsRNA triggers 14, 15, 16 and 17 asdescribed in Example 10. The seeds are germinated on paper and theseedlings is harvested at 7, 14 and 30 days post treatment and total RNAis extracted from whole seedlings, leaves or roots of the germinatedseeds. cDNA is prepared using oligo-dT primers and the expression levelsof TAS3, ARF3 and ARF4 are determined by real-time PCR with SYBR Green(Quanta BioSciences). ARF3 and ARF4 genes are regulated by the TAS3system and their expression is predicted to decrease following triggertreatment.

Although the disclosure has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present disclosure. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

1. An isolated double-stranded RNA (dsRNA) molecule comprising (a) afirst RNA strand having at least one antisense RNA sequence forsuppressing expression of a target gene of interest in a plant or aphytopathogen of a plant, a first heterologous smRNA-binding sequencefor binding to a first small RNA (smRNA) expressed in said plant orphytopathogen, and a helicase-binding sequence comprising the helicasebinding site of SEQ ID NO: 14; and (b) a second RNA strand that is areverse complement of said first RNA strand.
 2. The isolated dsRNAmolecule of claim 1, wherein said first smRNA comprises a nucleic acidsequence wherein said nucleic acid sequence shares between 100% and 90%sequence identity to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs:1 to 288, and complements thereof. 3.-5.(canceled)
 6. The isolated dsRNA molecule of claim 1, wherein said firstRNA strand further comprises a second heterologous smRNA-bindingsequence for binding a second smRNA expressed in said plant orphytopathogen, and said first heterologous smRNA-binding sequence andsaid second heterologous smRNA-binding sequence flank said at least oneantisense RNA sequence.
 7. The isolated dsRNA molecule of claim 6,wherein said first smRNA and said second smRNA comprise a nucleic acidsequence having at least 90% sequence identity to a nucleic acidsequence selected from the group consisting of SEQ ID NOs:1 to 288, andcomplements thereof.
 8. The isolated dsRNA molecule of claim 6, whereinsaid second heterologous smRNA-binding sequence is the complement ofsaid second smRNA.
 9. The isolated dsRNA molecule of claim 6, whereinsaid second smRNA is identical to said first smRNA.
 10. The isolateddsRNA molecule of claim 6, wherein said second smRNA is non-identical tosaid first smRNA.
 11. The isolated dsRNA molecule of claim 6, whereinsaid first heterologous smRNA-binding sequence and said secondheterologous smRNA-binding sequence comprise a nucleotide sequenceselected from the group consisting of: a direct sequence of said firstsmRNA and a direct sequence of said second smRNA; a reverse complementof said first smRNA and a direct sequence of said second smRNA; areverse complement of said first smRNA and a reverse complement of saidsecond smRNA; a direct sequence of said first smRNA and a reversecomplement of said second smRNA; a direct sequence of said first smRNAand further comprising a mutation rendering it resistant to cleavage anda direct sequence of said second smRNA; a reverse complement of saidfirst smRNA and further comprising a mutation rendering it resistant tocleavage and a direct sequence of said second smRNA; a reversecomplement of said first smRNA and further comprising a mutationrendering it resistant to cleavage and a reverse complement of saidsecond smRNA; a direct sequence of said first smRNA and furthercomprising a mutation rendering it resistant to cleavage and a reversecomplement of said second smRNA; a direct sequence of said first smRNAand a direct sequence of said second smRNA and further comprising amutation rendering it resistant to cleavage; a reverse complement ofsaid first smRNA and a direct sequence of said second smRNA and furthercomprising a mutation rendering it resistant to cleavage; a reversecomplement of said first smRNA and a reverse complement of said secondsmRNA and further comprising a mutation rendering it resistant tocleavage; a direct sequence of said first smRNA and a reverse complementof said second smRNA and further comprising a mutation rendering itresistant to cleavage; a direct sequence of said first smRNA and furthercomprising a mutation rendering it resistant to cleavage and a directsequence of said second smRNA and further comprising a mutationrendering it resistant to cleavage; a reverse complement of said firstsmRNA and further comprising a mutation rendering it resistant tocleavage and a direct sequence of said second smRNA and furthercomprising a mutation rendering it resistant to cleavage; a reversecomplement of said first smRNA and further comprising a mutationrendering it resistant to cleavage and a reverse complement of saidsecond smRNA and further comprising a mutation rendering it resistant tocleavage; and a direct sequence of said first smRNA and furthercomprising a mutation rendering it resistant to cleavage and a reversecomplement of said second smRNA and further comprising a mutationrendering it resistant to cleavage. 12.-14. (canceled)
 15. The isolateddsRNA molecule of claim 1, wherein said first smRNA has a nucleotidesequence selected from the group consisting of an RNA sequence of amicroRNA (miRNA) and an RNA sequence of an siRNA.
 16. The isolated dsRNAmolecule of claim 6, wherein said first smRNA has a nucleotide sequenceselected from the group consisting of an RNA sequence of a miRNA and anRNA sequence of an siRNA, and said second smRNA has a nucleotidesequence selected from the group consisting of an RNA sequence of amiRNA and an RNA sequence of an siRNA.
 17. (canceled)
 18. The isolateddsRNA molecule of claim 1, wherein said first smRNA is a miRNA.
 19. Theisolated dsRNA molecule of claim 6, wherein said first smRNA is a miRNAand said second smRNA is a miRNA.
 20. (canceled)
 21. The isolated dsRNAmolecule of claim 1, wherein said first smRNA is a miRNA selected fromthe group consisting of miR390, miR161.1, miR168, miR393, miR828, andmiR173.
 22. The isolated dsRNA molecule of claim 6, wherein said firstsmRNA is a miRNA selected from the group consisting of miR390, miR161.1,miR168, miR393, miR828, and miR173. 23.-48. (canceled)
 49. A method ofsuppressing gene expression in a plant or a phytopathogen of the plantcomprising: a. contacting a seed with an isolated double-stranded RNA(dsRNA) molecule of claim 1 under conditions which allow penetration ofsaid dsRNA molecule into said seed, thereby introducing said dsRNAmolecule into said seed; and optionally b. generating a plant of saidseed.
 50. The method of claim 49, wherein said dsRNA molecule penetratesa cell of said seed selected from the group consisting of an endospermcell, an embryo cell, and combinations thereof. 51.-69. (canceled) 70.An isolated double-stranded RNA (dsRNA) molecule comprising: a first RNAstrand having a nucleic acid sequence comprising in a sequential orderfrom 5′ to 3′; an endovirus 5′ UTR sequence; an endovirus RNA DependentRNA Polymerase (RDRP) coding sequence; a multiple cloning site; anendovirus 3′ UTR sequence; and a second RNA strand that is a reversecomplement of said first RNA strand.
 71. An isolated dsRNA moleculecomprising: a first RNA strand having a nucleic acid sequence comprisingin a sequential order from 5′ to 3′; an endovirus 5′ untranslated region(UTR) sequence; an endovirus RNA Dependent RNA Polymerase (RDRP) codingsequence; an antisense nucleic acid sequence for regulating a targetgene; an endovirus 3′ UTR sequence; and a second RNA strand that is areverse complement of said first RNA strand.
 72. The isolated dsRNAmolecule of claim 70, wherein said endovirus 5′ UTR sequence, saidendovirus RNA Dependent RNA Polymerase (RDRP) coding sequence and saidendovirus 3′ UTR sequence are capable of autonomous replication whenintroduced into a plant cell.
 73. The isolated dsRNA molecule of claim71, wherein said endovirus 5′ UTR sequence, said endovirus RNA DependentRNA Polymerase (RDRP) coding sequence and said endovirus 3′ UTR sequenceare capable of autonomous replication when introduced into a plant cell.74.-113. (canceled)